miR - 15a expression determined by qRT - PCR in P493 - 6 cells treated with tetracycline for 3 days ( + Tet ) to suppress MYC expression or in untreated ( - Tet ) cells ( mean ± st . dev . , n = 3 technical replicates ) .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).


Immunoblots showing expression levels of TSC1 , S6K / P - S6K and α - tubulin in P493 - 6 and HEK293T cells overexpressing miR - 15a or a control miRNA . At the right miR - 15a overexpression was determined by qRT - PCR in P493 - 6 and HEK293T cells ( mean ± st . dev . , n = 3 technical replicates ) .

B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O

Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).


Immunoblot showing TSC1 expression , S6K / P - S6K and α ‑ tubulin in MCF - 7 cells transfected with LNA modified anti - sense miR - 15a oligos or control LNAs . At the right , successful miR - 15a knockdown determined by qRT - PCR in MCF - 7 cells is shown ( mean ± st . dev . , n = 3 technical replicates ) .

B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O

Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).


Relative luciferase activities ( RLU ) derived from reporter constructs containing TSC1 - 3 ´ UTR wild type sequences or with mutated miR - 15a seed sequence binding site on co - transfection with miR - 15a in MCF - 7 cells ( mean ± st . dev . , n = 3 biological replicates ) . The drawing at the left shows the TSC1 - 3 ' UTR with the position of the miR - 15a / 16 - 1 seed sequence and used mutation indicated .

O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.


Rate of oxygen consumption in P493 - 6 ( - Tet ) cells ectopically expressing miR - 15a or control - miRNA vector , under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated ( mean ± st . dev . , n = 8 biological replicates ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).


A , Cell viability of NS , GBM , and astrocyte cell lines measured 72 h after irradiation ( fold vs . non - irradiated cells , ctrl ) . * : one - way ANOVA , P = 0 . 0001 .

O O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Cell viability of NS, GBM, and astrocyte cell lines measured 72 h after irradiation (fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.0001.


B , Cell viability of BT308NS and their pseudo - differentiated progeny ( BT308 diff ) measured 24 h after irradiation ( fold vs . non - irradiated cells , ctrl ) . * : t - test , P = 0 . 0006 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Cell viability of BT308NS and their pseudo-differentiated progeny (BT308 diff) measured 24 h after irradiation (fold vs. non-irradiated cells, ctrl). *: t-test, P=0.0006.


C , Flow cytometry analysis of phosphorylated histone H2AX ( H2AX ) at the indicated time points after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . * : one - way ANOVA , P = 0 . 03 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) at the indicated time points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.03.


D , Neutral comet assay performed 24 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Comet tail ' s length is proportional to DSB extent .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O

D, Neutral comet assay performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.


E , Western blot showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 after IR ( 5 Gy ) . Total ATM protein is also shown . Vinculin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

E, Western blot showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 after IR (5 Gy). Total ATM protein is also shown. Vinculin was used as loading control.


F , qPCR of RAD51 expression measured in NS , BT308 diff , and astrocytes at the indicated time - points after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . Data are represented as mean ± SEM .

O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, qPCR of RAD51 expression measured in NS, BT308 diff, and astrocytes at the indicated time-points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). Data are represented as mean ± SEM.


A , Flow cytometry analysis of PKH - 26 staining in irradiated BT308NS , 24 h after the last irradiation . The percentage of PKH - 26pos cells is indicated . Ctrl : non - irradiated cells . The mean fluorescence intensity ( MFI ) was as follow : ctrl , 58 . 21 ; 1 . 5 Gy × 4 , 69 . 35 ; 2 Gy × 3 , 68 . 20 ; 5 Gy , 60 . 29 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Flow cytometry analysis of PKH-26 staining in irradiated BT308NS, 24 h after the last irradiation. The percentage of PKH-26pos cells is indicated. Ctrl: non-irradiated cells. The mean fluorescence intensity (MFI) was as follow: ctrl, 58.21; 1.5 Gy × 4, 69.35; 2 Gy × 3, 68.20; 5 Gy, 60.29.


B , qPCR of SOX2 expression measured in NS 6 h after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 02 .

O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, qPCR of SOX2 expression measured in NS 6 h after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.02.


C , LDA ( sphere forming ) measuring the enrichment of GSCs after IR ( 5 Gy ) . Ctrl : non - irradiated cells * : 2 test , p < 0 . 05 .

O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

C, LDA (sphere forming) measuring the enrichment of GSCs after IR (5 Gy). Ctrl: non-irradiated cells *: 2 test, p<0.05.


E , Tumor incidence in the LDA performed in p3 mice , as described in D .

O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O

E, Tumor incidence in the LDA performed in p3 mice, as described in D.


F , Histogram showing the in vivo GSC frequency measured by LDA in p3 tumors . GSC frequency ( 95 % CI ) was as follows : NS - ctrl , 1 / 258 . 2 ( 97 . 67 - 683 . 9 ) ; NS - IR 1 / 22 . 1 ( 8 . 05 - 62 . 6 ) . * : 2 test , P = 1 . 6 × 10 - 4 .

O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-TISSUE O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Histogram showing the in vivo GSC frequency measured by LDA in p3 tumors. GSC frequency (95% CI) was as follows: NS-ctrl, 1/258.2 (97.67-683.9); NS-IR 1/22.1 (8.05-62.6). *: 2 test, P=1.6×10-4.


G , LDA ( sphere forming ) measuring the in vitro frequency of GSCs in cells derived from p3 tumors . * : 2 test , P = 0 . 0006 .

O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O

G, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from p3 tumors. *: 2 test, P=0.0006.


H , LDA ( sphere forming ) measuring the in vitro frequency of GSCs in cells derived from intracranial tumors generated by BT463NS , and irradiated in vivo ( 2 Gy × 3 days ) ( n = 3 ) . Ctrl : non - irradiated tumors . * : 2 test , P = 2 . 04 × 10 - 11 . Data are represented as mean ± CI or ± SEM in B .

O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from intracranial tumors generated by BT463NS, and irradiated in vivo (2 Gy × 3 days) (n = 3). Ctrl: non-irradiated tumors. *: 2 test, P=2.04×10-11. Data are represented as mean ± CI or ± SEM in B.


B , LDA ( sphere forming ) measuring the GSC frequency after IR ( 5 Gy ) in METhigh and METneg subpopulations sorted from BT308NS . * : 2 test , P = 0 . 001 .

O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

B, LDA (sphere forming) measuring the GSC frequency after IR (5 Gy) in METhigh and METneg subpopulations sorted from BT308NS. *: 2 test, P=0.001.


C , Flow cytometry analysis of MET in irradiated BT308NS , 24 h after the last irradiation ( 2 Gy × 3 days or a single dose of 5 Gy ) . Dotted lines : threshold to define the percentage of MET - expressing cells . Ctrl : non - irradiated cells . The MFI was as follows : ctrl , 18 ; 2 Gy × 3 , 22 . 77 ; 5 Gy , 19 . 49 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Flow cytometry analysis of MET in irradiated BT308NS, 24 h after the last irradiation (2 Gy × 3 days or a single dose of 5 Gy). Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells. The MFI was as follows: ctrl, 18; 2 Gy × 3, 22.77; 5 Gy, 19.49.


D , Flow cytometry analysis of MET in BT308NS weekly treated with IR ( 2 Gy ) for 6 weeks . Dotted lines : threshold to define the percentage of MET - expressing cells . Ctrl : non - irradiated cells .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

D, Flow cytometry analysis of MET in BT308NS weekly treated with IR (2 Gy) for 6 weeks. Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells.


E , Representative immunofluorescence staining of MET ( red ) on tumors generated by subcutaneous injection of BT308NS , irradiated ( 2 Gy × 3 ) and explanted 72 h after the last irradiation . Nuclei are counterstained with DAPI ( blue ) . Ctrl : non - irradiatedtumors . Scale bar , 10 µm ( 63 × magnification ) . F left , Quantification of the percentage of MET - expressing cells in tumor sections represented in ( E ) ( n = 12 HPF / group ) . HPF : high - power field . * : t - test , P = 0 . 001 . F right , Quantification of MET mean fluorescence intensity in tumor sections represented in ( E ) ( n = 12 HPF / group , fold vs . non - irradiated cells , ctrl ) . * : t - test , P = 0 . 00016 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, Representative immunofluorescence staining of MET (red) on tumors generated by subcutaneous injection of BT308NS, irradiated (2 Gy × 3) and explanted 72 h after the last irradiation. Nuclei are counterstained with DAPI (blue). Ctrl: non-irradiatedtumors. Scale bar, 10 µm (63× magnification). F left, Quantification of the percentage of MET-expressing cells in tumor sections represented in (E) (n = 12 HPF/group). HPF: high-power field. *: t-test, P=0.001. F right, Quantification of MET mean fluorescence intensity in tumor sections represented in (E) (n = 12 HPF/group, fold vs. non-irradiated cells, ctrl). *: t-test, P=0.00016.


G , representative METimmunohistochemistry staining of matched primary and recurrent tumors . Scale bar , 50 µm ( 40 × magnification ) . H , Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors : negative , 0 - 5 % ; low , 6 - 29 % ; moderate , 30 - 69 % ; high , 70 - 100 % ( see also Appendix Table 4 ) . I , Dot plot representing the MET cumulative score ( MET positive cell score + MET staining intensity score ) in 19 primary and recurrent tumors ( see Appendix Table 4 ) . Wilcoxon , p < 0 . 02 . Data are represented as mean ± SEM or ± CI in B .

O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, representative METimmunohistochemistry staining of matched primary and recurrent tumors. Scale bar, 50 µm (40× magnification). H, Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors: negative, 0-5%; low, 6-29%; moderate, 30-69%; high, 70-100% (see also Appendix Table 4). I, Dot plot representing the MET cumulative score (MET positive cell score + MET staining intensity score) in 19 primary and recurrent tumors (see Appendix Table 4). Wilcoxon, p<0.02. Data are represented as mean ± SEM or ± CI in B.


A , Flow - cytometry analysis of Annexin V / DAPI incorporation and MET expression in BT308NS 36 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Blue : MET - negative cells . Red : MET - expressing cells .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

A, Flow-cytometry analysis of Annexin V/DAPI incorporation and MET expression in BT308NS 36 h after IR (5 Gy). Ctrl: non-irradiated cells. Blue: MET-negative cells. Red: MET-expressing cells.


B , Western Blot of METneg and METhigh subpopulations sorted from BT452NS , showing activation of PARP ( cleaved PARP ) and Caspase 3 ( cleaved casp - 3 ) after IR ( 5 Gy ) . Vinculin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O

B, Western Blot of METneg and METhigh subpopulations sorted from BT452NS, showing activation of PARP (cleaved PARP) and Caspase 3 (cleaved casp-3) after IR (5 Gy). Vinculin was used as loading control.


C , Cell viability of METhigh and METneg subpopulations sorted from BT308NS , measured 96 h after IR ( 1 - 10 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 001 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Cell viability of METhigh and METneg subpopulations sorted from BT308NS, measured 96 h after IR (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.001.


D , Clonogenic assay with METhigh and METneg subpopulations sorted from BT308NS and irradiated ( 1 - 10 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 05 .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Clonogenic assay with METhigh and METneg subpopulations sorted from BT308NS and irradiated (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.05.


E , Neutral comet assay in METhigh and METneg subpopulations sorted from BT308NS , performed 24 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Comet tail ' s length is proportional to DSB extent .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O

E, Neutral comet assay in METhigh and METneg subpopulations sorted from BT308NS, performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.


F , Western Blot of METneg and METhigh subpopulations sorted from BT308NS , showing constitutive ( ctrl ) and / or IR - induced phosphorylation of ATM ( pATM ) , Chk2 ( pChk2 ) and H2AX ( H2AX ) , and accumulation of RAD51 , 24 h after IR ( 5 Gy ) . Total ATM protein is also shown . β - actin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

F, Western Blot of METneg and METhigh subpopulations sorted from BT308NS, showing constitutive (ctrl) and/or IR-induced phosphorylation of ATM (pATM), Chk2 (pChk2) and H2AX (H2AX), and accumulation of RAD51, 24 h after IR (5 Gy). Total ATM protein is also shown. β-actin was used as loading control.


G , qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR ( 5 Gy , fold vs . non - irradiated control cells , ctrl ) . * : t - test , P = 0 . 02 . Data are represented as mean ± SEM .

O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR (5 Gy, fold vs. non-irradiated control cells, ctrl). *: t-test, P=0.02. Data are represented as mean ± SEM.


A , Cell viability of BT308NS , measured 48 h after IR ( 2 or 5 Gy ) in the absence ( mock ) or in the presence of 10 ng / ml HGF ( fold vs . non - irradiated mock treated cells , mock ) . Ctrl : non - irradiated cells . * : t - test ( 5 Gy + HGF ) vs . ( 5 Gy ) , p < 0 . 0001 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

A, Cell viability of BT308NS, measured 48 h after IR (2 or 5 Gy) in the absence (mock) or in the presence of 10 ng/ml HGF (fold vs. non-irradiated mock treated cells, mock). Ctrl: non-irradiated cells. *: t-test (5 Gy + HGF) vs. (5 Gy), p<0.0001.


B , Flow - cytometry analysis of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in the absence ( mock ) or in the presence of HGF . Histograms show the percentage of Annexin V - positive cells 48 h after IR . Ctrl : non - irradiated cells .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

B, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (mock) or in the presence of HGF. Histograms show the percentage of Annexin V-positive cells 48 h after IR. Ctrl: non-irradiated cells.


C , Cell viability of BT308NS , irradiated in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) , and analyzed at the indicated time points . Vehicle : non - irradiated cells ( fold vs . vehicle - treated cells at time 0 , mock ) . * : t - test , p < 0 . 01 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Cell viability of BT308NS, irradiated in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM), and analyzed at the indicated time points. Vehicle: non-irradiated cells (fold vs. vehicle-treated cells at time 0, mock). *: t-test, p<0.01.


D , Cell viability as in C , measured 5 days after IR ( 1 - 10 Gy , fold vs . vehicle - treated cells , mock ) . * : t - test , p < 0 . 03 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Cell viability as in C, measured 5 days after IR (1-10 Gy, fold vs. vehicle-treated cells, mock). *: t-test, p<0.03.


E , Flow - cytometry analysis of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in the absence ( vehicle ) or in the presence ( JNJ ) of JNJ38877605 . Histograms show the percentage of Annexin V - positive cells 36 h after IR . Ctrl : non - irradiated cells . * : t - test ( 5 Gy + JNJ ) vs . ( 5 Gy ) , P = 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

E, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Histograms show the percentage of Annexin V-positive cells 36 h after IR. Ctrl: non-irradiated cells. *: t-test (5 Gy + JNJ) vs. (5 Gy), P=0.01.


F , Western blot of BT308NS showing caspase 3 activation ( cleaved casp - 3 ) , at the indicated time points after IR in the absence ( - ) or in the presence ( + ) of JNJ38877605 . Vinculin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O

F, Western blot of BT308NS showing caspase 3 activation (cleaved casp-3), at the indicated time points after IR in the absence (-) or in the presence (+) of JNJ38877605. Vinculin was used as loading control.


G , Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS , irradiated ( 5 Gy ) in the absence ( vehicle ) or in the presence of MET inhibitors ( JNJ38877605 and Crizotinib , 500 nM ) . Ctrl : non - irradiated cells . * : t - test , p < 0 . 0004 .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

G, Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS, irradiated (5 Gy) in the absence (vehicle) or in the presence of MET inhibitors (JNJ38877605 and Crizotinib, 500 nM). Ctrl: non-irradiated cells. *: t-test, p<0.0004.


H , LDA ( sphere forming assay ) with METneg and METhigh subpopulations sorted from BT308NS , measuring GSC frequency after irradiation in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated cells . * : 2 test , P = 0 . 0002 .

O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

H, LDA (sphere forming assay) with METneg and METhigh subpopulations sorted from BT308NS, measuring GSC frequency after irradiation in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. *: 2 test, P=0.0002.


I , Flow cytometry analysis of phosphorylated histone H2AX ( H2AX ) in the whole BT308NS at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 ( fold vs . non - irradiated cells at time 0 , mock ) . * : t - test , p < 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

I, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) in the whole BT308NS at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605 (fold vs. non-irradiated cells at time 0, mock). *: t-test, p<0.05.


J , Neutral comet assay with BT308NS performed 24 h after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated cells . Comet tail ' s length is proportional to DSB extent . Data are represented as mean ± SEM or ± CI in H .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

J, Neutral comet assay with BT308NS performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. Comet tail's length is proportional to DSB extent. Data are represented as mean ± SEM or ± CI in H.


A , Western blot of BT463NS showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) . Total ATM is also shown . H3 was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

A, Western blot of BT463NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). Total ATM is also shown. H3 was used as loading control.


B , Western blot of BT308NS showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) . Total ATM is also shown . β - actin was used as loading control . Veh : non - irradiated vehicle - treated cells . C , Densitometric analysis of ATM phosphorylation shown in ( B ) . * : t - test , p < 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

B, Western blot of BT308NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). Total ATM is also shown. β-actin was used as loading control. Veh: non-irradiated vehicle-treated cells. C, Densitometric analysis of ATM phosphorylation shown in (B). *: t-test, p<0.01.


D , Western blot of BT308NS showing phosphorylation of ATM , Chk2 , and AKT ( pAKT ) , and accumulation of RAD51 24 h after IR ( 5 Gy ) in the absence ( vehicle , veh ) or in the presence of MAPK ( PD98059 , 20 M ) , AKT ( Ly294002 , 20 M ) , MET ( JNJ38877605 ) , or ATM ( CGK733 , 10 M ) inhibitors . Total ATM and AKT are also shown . β - actin was used as loading control . Ctrl : non - irradiated cells .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

D, Western blot of BT308NS showing phosphorylation of ATM, Chk2, and AKT (pAKT), and accumulation of RAD51 24 h after IR (5 Gy) in the absence (vehicle, veh) or in the presence of MAPK (PD98059, 20 M), AKT (Ly294002, 20 M), MET (JNJ38877605), or ATM (CGK733, 10 M) inhibitors. Total ATM and AKT are also shown. β-actin was used as loading control. Ctrl: non-irradiated cells.


E , Western blot of BT463NS showing phosphorylation of ATM , Chk2 , and Aurora Kinase A ( pAurora A ) at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + Ly ) of the AKT inhibitor Ly294002 . Total ATM and Aurora Kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O

E, Western blot of BT463NS showing phosphorylation of ATM, Chk2, and Aurora Kinase A (pAurora A) at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + Ly) of the AKT inhibitor Ly294002. Total ATM and Aurora Kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


F , Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Total Aurora kinase A is also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O

F, Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Total Aurora kinase A is also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


G , Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF . Total Aurora kinase A is also shown . H3 was used as loading control ( the same control applies to both panel A and G ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

G, Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF. Total Aurora kinase A is also shown. H3 was used as loading control (the same control applies to both panel A and G).


H , Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + MLN ) of the Aurora Kinase A inhibitor MLN8237 ( 20 M ) . Total ATM and Aurora kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O

H, Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + MLN) of the Aurora Kinase A inhibitor MLN8237 (20 M). Total ATM and Aurora kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.


I , Cell viability of BT308NS , measured 72 h after IR ( 5 Gy ) in the absence ( vehicle ) or in the presence of MET ( JNJ38877605 ) , Aurora Kinase A ( MLN8237 ) , or AKT ( Ly294002 ) inhibitors ( fold vs . non - irradiated vehicle - treated cells , mock ) . * : t - test ( 5 Gy + inhibitor ) vs . ( 5 Gy + vehicle ) , p < 0 . 01 .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I, Cell viability of BT308NS, measured 72 h after IR (5 Gy) in the absence (vehicle) or in the presence of MET (JNJ38877605), Aurora Kinase A (MLN8237), or AKT (Ly294002) inhibitors (fold vs. non-irradiated vehicle-treated cells, mock). *: t-test (5 Gy + inhibitor) vs. (5 Gy + vehicle), p<0.01.


A , Western blot of BT371NS showing p21 phosphorylation ( pp21Thr145 ) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

A, Western blot of BT371NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml).


B , Western blot of BT463NS showing p21 phosphorylation ( pp21Thr145 ) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O

B, Western blot of BT463NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM).


C , Western blot of BT308NS showing p21 localization in cytoplasmic / nuclear extracts at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions , respectively .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O

C, Western blot of BT308NS showing p21 localization in cytoplasmic/nuclear extracts at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions, respectively.


D , Representative immunofluorescence staining of p21 ( red ) on BT308NS cells 3 h after IR ( 5 Gy ) in the absence ( vehicle ) or in the presence ( JNJ ) of JNJ38877605 . Ctrl : non - irradiated cells . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 µm ( 63 × magnification ) . E , Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in ( D ) ( n = 10 HPF / group ) . HPF : high - power field . * : t - test ( 5 Gy + JNJ ) vs . ( ctrl or 5 Gy ) , p < 0 . 0001 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

D, Representative immunofluorescence staining of p21 (red) on BT308NS cells 3 h after IR (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Ctrl: non-irradiated cells. Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm (63× magnification). E, Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in (D) (n = 10 HPF/group). HPF: high-power field. *: t-test (5 Gy + JNJ) vs. (ctrl or 5 Gy), p<0.0001.


F , Cell cycle analysis of BT314NS , performed 24 h after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated cells . * : one - way ANOVA , P = 0 . 008 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

F, Cell cycle analysis of BT314NS, performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated cells. *: one-way ANOVA, P=0.008.


B right , Quantification of epi - fluorescent GFP signal from tumors represented in ( B , left ) ( n = 4 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 5 . 5 × 10 - 5 .

O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

B right, Quantification of epi-fluorescent GFP signal from tumors represented in (B, left) (n = 4/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=5.5×10-5.


C , Growth curves of tumors , generated by subcutaneous injection of BT308NS , irradiated in the absence ( 2 Gy × 3 days ) or in the presence ( combo ) of JNJ38877605 , which was administered for 15 days as indicated ( n = 7 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 0 . 0006 .

O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O

C, Growth curves of tumors, generated by subcutaneous injection of BT308NS, irradiated in the absence (2 Gy × 3 days) or in the presence (combo) of JNJ38877605, which was administered for 15 days as indicated (n = 7/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=0.0006.


D , Survival analysis of mice bearing tumors generated and treated as in C . Black dot : censored mouse . Log - rank ( Mantel - Cox ) test ( combo ) vs . ( 2 Gy × 3 days ) , P = 0 . 0019 .

O O B-EXP_ASSAY O O B-ORGANISM O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Survival analysis of mice bearing tumors generated and treated as in C. Black dot: censored mouse. Log-rank (Mantel-Cox) test (combo) vs. (2 Gy × 3 days), P=0.0019.


F , In vitro LDA ( sphere - forming ) measuring GSC frequencies as described in E ( n = 3 / condition ) . * : 2 test , p < 10 - 5 .

O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

F, In vitro LDA (sphere-forming) measuring GSC frequencies as described in E (n = 3/condition). *: 2 test, p<10-5.


G , Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E .

O O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O

G, Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E.


H , Histogram showing GSC frequencies measured by in vivo LDA , as described in E . * : 2 test , P = 5 . 47 × 10 - 6 . I , Table showing GSC frequencies measured by in vivo LDA , represented in H . Data are represented as mean ± SEM or ± CI in F , H , I .

O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

H, Histogram showing GSC frequencies measured by in vivo LDA, as described in E. *: 2 test, P=5.47×10-6. I, Table showing GSC frequencies measured by in vivo LDA, represented in H. Data are represented as mean ± SEM or ± CI in F,H,I.


( A , B ) Poly ( I : C ) treatment inhibits axonal growth at 3 DIV ( A ) and dendritic growth at 6 DIV ( B ) in WT neurons . Neurons were transfected with GFP at 1 or 4 DIV . One day later , neurons were treated with poly ( I : C ) for 24 h before harvest . ( C ) Axon and dendrite morphology of Tlr3 - / - neurons after poly ( I : C ) treatment . Data in A , B , C were analyzed by unpaired t - test . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 20μm in A and B .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B.


( D , E ) Neuronal morphology in saline - and poly ( I : C ) - treated Tlr3 + / + ; Thy1 - Yfp ( D ) and Tlr3 - / - ; Thy1 - Yfp ( E ) mouse brains . Total dendrite length , dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype . Data of dendrite length and tip number were analyzed by unpaired t - test . The Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . The numbers of analyzed neurons and mice are presented in each column . Mean values SEM are shown . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 50 μm in D and E .

O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM B-TISSUE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3-/-;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.


( A , B ) Quantitation of dendrite morphology of Trif mutant neurons ( A ) and Myd88 - / - neurons ( B ) after poly ( I : C ) stimulation . Trif mutant or Myd88 - / - neurons were transfected with a GFP construct at 4 DIV and treated with poly ( I : C ) one day later . Neuron morphology was analyzed at 6 DIV according to the GFP signal . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test .

O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88-/- neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88-/- neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.


( C ) MYD88 but not TRIF expression reduces dendrite outgrowth . HA - tagged MYD88 and TRIF were co - transfected with a GFP construct into cultured wild - type ( WT ) neurons at 4 DIV , and cell morphology was analyzed at 6 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by one - way ANOVA with Bonferroni ' s multiple comparison test .

O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni's multiple comparison test.


( D ) MYD88 knockdown , but not TRIF knockdown , robustly increases dendritic arbors and loses the response to poly ( I : C ) . Three DIV neurons were transfected with control shRNA ( shCtrl ) , MYD88 shRNA ( shMYD88 ) or TRIF shRNA ( shTRIF ) and poly ( I : C ) was applied into the culture at 5 DIV for 24 h before harvest . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * * * P < 0 . 0001 .

O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.


( A ) Coimmunoprecipitation of TLR3 and MYD88 . HEK293T cells were transfected with the indicated constructs . Twenty - four h later , immunoprecipitation ( IP ) was performed using HA antibody . The precipitates were immunoblotted ( IB ) with Myc and HA antibodies as indicated . EC , TLR3 ectodomain .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O

(A) Coimmunoprecipitation of TLR3 and MYD88. HEK293T cells were transfected with the indicated constructs. Twenty-four h later, immunoprecipitation (IP) was performed using HA antibody. The precipitates were immunoblotted (IB) with Myc and HA antibodies as indicated. EC, TLR3 ectodomain.


( B ) Poly ( I : C ) treatment increases the interaction between TLR3 and MYD88 . One day after transfection , cells were treated with saline or poly ( I : C ) for 30 min before harvesting .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

(B) Poly(I:C) treatment increases the interaction between TLR3 and MYD88. One day after transfection, cells were treated with saline or poly(I:C) for 30 min before harvesting.


( C ) Upper , schematic of MYD88 constructs . DD , death domain ; ID , intermediate domain , TIR , TIR domain . Lower , coimmunoprecipitation of TLR3 and the N - terminal region of MYD88 .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O

(C) Upper, schematic of MYD88 constructs. DD, death domain; ID, intermediate domain, TIR, TIR domain. Lower, coimmunoprecipitation of TLR3 and the N-terminal region of MYD88.


( D ) Full - length and N - terminal MYD88 inhibit dendrite outgrowth . Myd88 - / - neurons were transfected with control vector ( Ctrl ) , HA - tagged MYD88 ( MYD88 ) , N - terminal MYD88 ( MYD88 - N ) , or C - terminal MYD88 ( MYD88 - C ) with a GFP construct . Poly ( I : C ) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm .

O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Full-length and N-terminal MYD88 inhibit dendrite outgrowth. Myd88-/- neurons were transfected with control vector (Ctrl), HA-tagged MYD88 (MYD88), N-terminal MYD88 (MYD88-N), or C-terminal MYD88 (MYD88-C) with a GFP construct. Poly(I:C) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm.


( E ) Expression patterns of full - length and truncated fragments of MYD88 in Myd88 - / - neurons . Representative images are shown .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O

(E) Expression patterns of full-length and truncated fragments of MYD88 in Myd88-/- neurons. Representative images are shown.


( A ) qPCR analysis of proinflammatory cytokines Il - 6 , Tnfα , Il - 1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons . Data were analyzed by unpaired t - test . Il - 6 and Tnfα N = 8 ; Ifnβ and Il - 1β , N = 6 .

O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(A) qPCR analysis of proinflammatory cytokines Il-6, Tnfα, Il-1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons. Data were analyzed by unpaired t-test. Il-6 and Tnfα N=8; Ifnβ and Il-1β, N=6.


( B , C ) Poly ( I : C ) treatment inhibits dendrite outgrowth of Il - 6 - / - neurons ( B ) and Tnfα - / - neurons ( C ) . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(B, C) Poly(I:C) treatment inhibits dendrite outgrowth of Il-6-/- neurons (B) and Tnfα-/- neurons (C). Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.


( D ) Left , schematic of experiment using conditioned medium . Upper right , conditioned medium applied to WT neurons . Lower right , conditioned medium applied to Tlr3 - / - neurons . Dendrite morphology was analyzed one day after adding conditioned medium . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test . * P < 0 . 05 , * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Left, schematic of experiment using conditioned medium. Upper right, conditioned medium applied to WT neurons. Lower right, conditioned medium applied to Tlr3-/- neurons. Dendrite morphology was analyzed one day after adding conditioned medium. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. * P < 0.05, *** P < 0.0001.


At 4 DIV , cultured cortical neurons were transfected with indicated plasmids . Three days later , neurons were treated with 10 μg / ml poly ( I : C ) for 24 h . Neuronal morphology was monitored by GFP signals at 8 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * * * P < 0 . 0001 , ns : non - significant .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

At 4 DIV, cultured cortical neurons were transfected with indicated plasmids. Three days later, neurons were treated with 10 μg/ml poly(I:C) for 24 h. Neuronal morphology was monitored by GFP signals at 8 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001, ns: non-significant.


( A ) Expression of 12 neuropsychiatric disorder - related genes in saline - and poly ( I : C ) - treated WT neurons .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_TYPE O

(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons.


( B ) Expression of 9 neuropsychiatric disorder - related genes in poly ( I : C ) - treated Tlr3 - / - neurons . Relative expression levels compared with vehicle control are shown . In ( B - D ) , dashed lines indicate the level of saline control .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control.


( C , D ) Expression of Auts2 , Disc1 , Fmr1 , Pten and Ube3a in poly ( I : C ) - treated Myd88 - / - neurons ( C ) and poly ( I : C ) - treated P5 WT mouse brains ( D ) . In ( B - D ) , dashed lines indicate the level of saline control .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O

(C, D) Expression of Auts2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88-/- neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). In (B-D), dashed lines indicate the level of saline control.


( E ) DIV 5 WT neurons were treated with poly ( I : C ) for 24 h . The total neuronal lysates were subjected to immunoblotting with the indicated antibodies . VCP and β - actin were used as loading controls . The protein levels were normalized with β - actin . Data were analyzed by unpaired t - test . Numbers of experimental repeats ( N ) are shown in each column . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 .

O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.


( A ) Overexpression of WT DISC1 , but not the DISC1 L604F mutant , in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Overexpression of WT DISC1, but not the DISC1 L604F mutant, in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. * P < 0.05, ** P < 0.001, *** P < 0.0001.


( B - C ) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly ( I : C ) - triggered reduction of dendritic arborization . Vector control ( Ctrl ) ( B ) or Myc - DISC1 ( C ) was co - expressed with GFP in cortical layer 2 / 3 neurons in mouse brain . After poly ( I : C ) injection at P4 and P5 , the neuronal morphology was analyzed at P7 by tracing the GFP signals . The data of dendrite length and tip number were analyzed by unpaired t - test . The Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . The numbers of analyzed neurons collected from 3 - 4 mice of each group are indicated . Mean values SEM are shown . Scale bar , 30 μm . * P < 0 . 05 , * * * P < 0 . 0001 .

O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.


( A ) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly ( I : C ) ( pIC ) at 17 DIV for 24 h . Spine morphology was examined at 18 DIV . Three secondary dendrites of each neuron were selected to analyze the spine density , the width of the spine head and the spine length . Mean values SEM of representatives of three independent experiments are shown . For the saline group , 15 neurons / 45 dendrites were examined ; for the poly ( I : C ) group , 16 neurons / 48 dendrites were examined . Data were analyzed by unpaired t - test . Scale bar , 20 μm in right upper panel and 5 µm in lower left panel . * * * P < 0 . 0001 .

O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001.


( C , D ) Spine morphology of P21 Thy1 - Yfp ( C ) and Tlr3 - / - ; Thy1 - Yfp mice ( D ) after poly ( I : C ) stimulation at P4 and P5 . One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology . Three mice were analyzed for each group . Saline - treated Thy1 - Yfp , 44 neurons ; poly ( I : C ) - treated Thy1 - Yfp , 43 neurons ; saline - treated Tlr3 - / - ; Thy1 - Yfp , 46 neurons ; poly ( I : C ) - treated Tlr3 - / - ; Thy1 - Yfp , 50 neurons . Mean values SEM are shown . Data were analyzed by unpaired t - test . Scale bar , 5 μm . * * * P < 0 . 0001 .

O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-SUBCELLULAR O O O B-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3-/-; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3-/-;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3-/-;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.


The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps . ( top panel ) The voltage protocol used for the experiment . ( bottom panel ) Representative traces of currents elicited by voltage ramp pulses are shown . ( top panel ) . The red line represents the arithmetical difference between currents elicited by 500 - ms ( 2 ) and 2 - ms pulses ( 1 ) . B . The summary bar graph represents the mean values of the reversal potentials of currents . ( 9 cells from 3 mice , paired Student t - test ; data are presented as mean values ± s . e . m ) .

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps. (top panel) The voltage protocol used for the experiment. (bottom panel) Representative traces of currents elicited by voltage ramp pulses are shown. (top panel). The red line represents the arithmetical difference between currents elicited by 500-ms (2) and 2-ms pulses (1). B. The summary bar graph represents the mean values of the reversal potentials of currents. (9 cells from 3 mice, paired Student t-test; data are presented as mean values ± s.e.m).


Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations . Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl - and 0 . 2 mM EGTA ( top panel ) , 150 mM gluconate ( middle panel ) , or 2 mM BAPTA ( bottom panel ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O

Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations. Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl- and 0.2 mM EGTA (top panel), 150 mM gluconate (middle panel), or 2 mM BAPTA (bottom panel).


A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration ( the numbers on the right indicate the numbers of recorded cells , data are presented as mean values ± s . e . m . )

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration (the numbers on the right indicate the numbers of recorded cells, data are presented as mean values ± s.e.m.)


A bar graph of averaged values of the tail current amplitudes summarizing the results of ( c ) at 500 ms . ( The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice , unpaired Student t - test ; data are presented as mean values ± s . e . m . ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

A bar graph of averaged values of the tail current amplitudes summarizing the results of (c) at 500 ms. (The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice, unpaired Student t-test; data are presented as mean values ± s.e.m.).


A summary bar graph showing the quantitative real - time PCR data for TMEM16A , TMEM16B , and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains ( N = 3 , n = 3 each , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.).


RNAscope ISH showed an apparent expression of TMEM16A mRNA co - localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb . Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level .

O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O B-GENEPROD O O O B-TISSUE O O O O O O O O

RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level.


Immunohistochemical staining of 40 - µm coronal brain sections with anti - TMEM16A antibody . In the mHb , TMEM16A signals colocalize with the cholinergic marker ChAT ( arrows ) . High magnifications of the indicated areas are shown in the merged panel .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-TISSUE O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.


Immunohistochemical staining of 40 - µm coronal brain sections with an anti - TMEM16A antibody . The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls . TMEM16A colocalized with the cholinergic marker ChAT in the mHb . The specific deletion of TMEM16A in the mHb is evident .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD O B-ORGANISM O O O O O O B-ORGANISM O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O O B-TISSUE O O O

Immunohistochemical staining of 40-µm coronal brain sections with an anti-TMEM16A antibody. The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls. TMEM16A colocalized with the cholinergic marker ChAT in the mHb. The specific deletion of TMEM16A in the mHb is evident.


Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell - attached mode from TMEM16A cKO mice compared to the one from littermate controls . D . A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO ( 4 mice ) and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells from 4 mice each , paired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O O B-GENEPROD O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell-attached mode from TMEM16A cKO mice compared to the one from littermate controls. D. A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO (4 mice) and littermate controls (The numbers inside each bar indicate the numbers of recorded cells from 4 mice each, paired Student t-test, data are presented as mean values ± s.e.m.).


Representative averaged traces of sAPs of the mHb neurons with the pipette solution ( 40 mM Cl - and 0 . 5 mM EGTA ) from TMEM16A cKO mice and littermate controls .

O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-ORGANISM O B-ORGANISM O O

Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls.


Summary bar graphs showing the frequency , half - width , and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O B-GENEPROD O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Summary bar graphs showing the frequency, half-width, and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls (The numbers inside each bar indicate the numbers of recorded cells, unpaired Student t-test, data are presented as mean values ± s.e.m.).


Immunohistochemical images of the IPN region from control and TMEM16A cKO mice . The c - Fos signal was greatly reduced in TMEM16A cKO mice . Scale bar = 200 μm .

B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-GENEPROD O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O O

Immunohistochemical images of the IPN region from control and TMEM16A cKO mice. The c-Fos signal was greatly reduced in TMEM16A cKO mice. Scale bar = 200 μm.


A summary bar graph showing reduced c - Fos expression in the IPN in TMEM16A cKO mice . ( NCTL = 7 ( n = 42 ) , NcKO = 6 ( n = 36 ) , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A summary bar graph showing reduced c-Fos expression in the IPN in TMEM16A cKO mice. (NCTL=7 (n=42), NcKO=6 (n=36), unpaired Student t-test, data are presented as mean values ± s.e.m.).


Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls .

O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O B-TISSUE O O O O O B-GENEPROD O B-ORGANISM O O O O B-ORGANISM O O

Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls.


Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO ( cKO ) mice and littermate controls ( CTL ) . Data are presented as mean values ± s . e . m . ( paired Student t - test ) . The numbers inside each bar indicate the numbers of recorded cells .

O O O O O O O O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO (cKO) mice and littermate controls (CTL). Data are presented as mean values ± s.e.m. (paired Student t-test). The numbers inside each bar indicate the numbers of recorded cells.


In the EPM task , the time spent in the open arms was significantly decreased in cKO mice compared to that in the control ( CTL ) group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . B . In the LDB task , the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . C . In the PA task , the entry latency of cKO mice was significantly decreased compared to that of the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . . D . Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group . The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In the EPM task, the time spent in the open arms was significantly decreased in cKO mice compared to that in the control (CTL) group (nCTL=20, ncKO=12, unpaired Student t-test). B. In the LDB task, the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test). C. In the PA task, the entry latency of cKO mice was significantly decreased compared to that of the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test)..D. Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group. The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group (nCTL=20, ncKO=12, unpaired Student t-test).


In the 3 - chamber test , the cKO mice exhibited a decreased interest for the stranger mouse ( S1 ) against objects ( O ) compared to the CTL group ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . F . When cKO mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to that of the CTL group . The preference index showed the decreased sociability of the cKO group in both sessions ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . Data information : All data are presented as mean values ± s . e . m .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In the 3-chamber test, the cKO mice exhibited a decreased interest for the stranger mouse (S1) against objects (O) compared to the CTL group (nCTL=12, ncKO=14, unpaired Student t-test). F. When cKO mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to that of the CTL group. The preference index showed the decreased sociability of the cKO group in both sessions (nCTL=12, ncKO=14, unpaired Student t-test). Data information: All data are presented as mean values ± s.e.m.


Representative traces showing that the spontaneous firing of mHb neurons from ChAT ( + ) - cre : : hM4D ( Gi ) mice were reduced when CNO ( 10 μM ) was applied in the ACSF .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O

Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF.


A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV - hM4Di - mCherry before and during CNO treatment ( The numbers inside each bar indicate the numbers of recorded cells ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells).


In the EPM task , the time spent in the open arms of CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that of the PBS - treated controls ( CTL ) . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . E . In the LDB task , the time spent in the light zone by CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that spent by the PBS - treated control mice . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . F . In the PA task , the entry latency of CNO - treated mice was significantly decreased compared to that of the CTL group . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . G . Nestlet shredding behaviors of the CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were also decreased . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) .

O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;).


In the 3 - chamber test , the subject mouse of the PBS - treated mice and the CNO - treated mice explored freely the three test chambers . The time spent in each chamber during two sessions was measured . The CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice exhibited a decreased interest for a stranger mouse ( S1 ) against objects ( O ) compared to the PBS - treated group ( left two panels ) ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . I . When CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to the PBS - treated control group ( right two panels ) . The preference index showed decreased sociability of the CNO - treated group in both sessions ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).


Cell surface H2 - Kb levels of wild type and TAP1 - / - BMDC transduced with control vector or a vector expressing hβ2m were analyzed .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O

Cell surface H2-Kb levels of wild type and TAP1-/- BMDC transduced with control vector or a vector expressing hβ2m were analyzed.


Cross - presentation of OVA by wild type and TAP1 - / - BMDC expressing hβ2m were compared to control BMDC , by measuring IL - 2 production by a Kb - SIINFEKL specific T - cell hybridoma ( B3Z ) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA - coated latex beads for 6hr , prior to fixation .

O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O

Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.


The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL - 2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV - OVA infected WT and TAP1 - / - BMDC transduced with control vector or hβ2m .

O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O

The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA infected WT and TAP1-/- BMDC transduced with control vector or hβ2m.


Surface H2 - Kb levels of wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed by flow cytometry .

O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

Surface H2-Kb levels of wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry.


Cross - presentation of OVA by wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed and IL - 2 release is

O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

Cross-presentation of OVA by wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed and IL-2 release is shown


TAP1 - / - BMDC transduced with vector control ( A ) or hβ2m ( C ) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb - binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry . Representative plots are shown ( A , C ) . The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin , normalized to that on cell incubated without Epoxomicin ( n = 3 ) ( B , D ) .

B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).


The effect of Epoxomicin on cross - presentation of OVA - coated beads by TAP1 - / - BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was

O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O

The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or US6 were analyzed for cross - presentation of OVA using B3Z cells . The effects of US6 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing US6 ( A ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or ICP47 ( B ) , were analyzed for cross - presentation of OVA using B3Z cells . The effects of ICP47 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing ICP47 ( B ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab7ADN ( E ) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.