( O ) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC - tag , MYC - RASSF1A ( 1 - 340 ) or MYC - RASSF1A ( 1 - 288 ) . 24 h later cells were transfected with I - PpoI mRNA and stained for MYC expression and ATM - pS1981 .

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

(O) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC-tag, MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and stained for MYC expression and ATM-pS1981.


( P ) Quantification of cells with ATM - pS1981 positive nucleolar caps in ( O ) . Error bars represent standard deviation and derive from 3 independent experiments .

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

(P) Quantification of cells with ATM-pS1981 positive nucleolar caps in (O). Error bars represent standard deviation and derive from 3 independent experiments.


( A ) HeLa cells were pre - treated with the ATMi or ATRi prior to transfection with I - PpoI mRNA . Cells were stained for RPA and quantified . Error bars represent standard deviation and derive from 3 independent experiments . ( B ) HeLa cells were pre - treated with the ATMi or ATRi prior to transfection with I - PpoI mRNA . Cells were stained for RAD51 and quantified . Error bars represent standard deviation and derive from 3 independent experiments .

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

(A) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RPA and quantified. Error bars represent standard deviation and derive from 3 independent experiments. (B) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RAD51 and quantified. Error bars represent standard deviation and derive from 3 independent experiments.


( C - F ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or two different siRNAs against RASSF1A ( siR1A _ 1 and siR1A _ 2 ) and 48 h later cells were transfected with I - PpoI mRNA to induce rDNA DSBs . 6 h post mRNA transfection , cells were stained for V5 to identify I - PpoI transfected cells and the other indicated antibodies . Representative images ( C , E ) and quantification of staining ( D , F ) for the indicated antibodies are shown . Error bars represent standard deviation and derive from 3 independent experiments .

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

(C-F) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (C, E) and quantification of staining (D, F) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.


( G ) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies .

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

(G) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies.


( H ) Cells were treated with siLUCIFERASE ( siLUC ) or si53BP1 and 48 h later transfected with I - PpoI mRNA and stained for RPA . ( I ) Quantification of ( H ) . 107 values were analysed in siLUC and 90 values were analysed in si53BP1 .

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 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 O

(H) Cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for RPA. (I) Quantification of (H). 107 values were analysed in siLUC and 90 values were analysed in si53BP1.


( J ) U2OS cells were treated with siLUCIFERASE ( siLUC ) , siRASSF1A ( siR1A ) or si53BP1 , treated with 10 μM BrdU for 24 h prior to I - PpoI mRNA transfection . 6 h post I - PpoI treated cells were pre - extracted , fixed and stained for BrdU under non denaturing conditions to visualize ssDNA . ( K ) Quantification of ( J ) . Fold change of nuclear BrdU signal relative to siLUC is shown . Error bars represent standard deviation and derive from 3 independent experiments .

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

(J) U2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 μM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non denaturing conditions to visualize ssDNA. (K) Quantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from 3 independent experiments.


( L ) HeLa cells were transfected with I - PpoI endonuclease and stained for MST2 and γH2AX . Fluorescence intensity profiles of MST2 ( green ) and γH2AX ( red ) signals across the HeLa nuclei are shown . Position of line scan indicated by the yellow line .

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

(L) HeLa cells were transfected with I-PpoI endonuclease and stained for MST2 and γH2AX. Fluorescence intensity profiles of MST2 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( M ) Validation of the MST2 siRNA with western blot .

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

(M) Validation of the MST2 siRNA with western blot.


( N ) HeLa cells treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h later transfected with I - PpoI mRNA . Cells were treated with 5 - EU for 30 min prior to fixation and assessed for incorporation . ( O ) Quantification of 5 - EU intensity in siLUCIFERASE ( siLUC ) or siMST2 treated cells transfected with I - PpoI mRNA . Middle line represents the median and the boxes 25th and 75th percentiles . The whiskers mark the smallest and largest values . 100 values were analyzed in each condition .

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

(N) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation. (O) Quantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.


( P ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h post transfection cells were transfected with I - PpoI mRNA . 6 h post transfection cells were fixed and stained for RASSF1A ( R1A ) and γH2AX . ( Q ) Quantification of ( P ) . Error bars represent standard deviation and derive from 3 independent experiments .

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

(P) HeLa cells were treated with siLUCIFERASE (siLUC) or siMST2 and 48 h post transfection cells were transfected with I-PpoI mRNA. 6 h post transfection cells were fixed and stained for RASSF1A (R1A) and γH2AX. (Q) Quantification of (P). Error bars represent standard deviation and derive from 3 independent experiments.


( R - W ) HeLa cells treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h later transfected with I - PpoI mRNA . Cells were stained for ATM - pS1981 ( R ) , KAP1 - pS824 ( T ) and RPA ( V ) and quantified ( S , U , W ) . 108 values in ( S ) , 95 values in ( U ) and 100 values in ( W ) were analysed .

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

(R-W) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were stained for ATM-pS1981 (R), KAP1-pS824 (T) and RPA (V) and quantified (S, U, W). 108 values in (S), 95 values in (U) and 100 values in (W) were analysed.


( Β ) Clonogenic survival analysis of HeLa cells treated with the indicated siRNAs and the indicated doses of Ionizing radiation ( γIR ) . Survival fraction was calculated . Error bars represent SEM and derive from 3 independent experiments .

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

(Β) Clonogenic survival analysis of HeLa cells treated with the indicated siRNAs and the indicated doses of Ionizing radiation (γIR). Survival fraction was calculated. Error bars represent SEM and derive from 3 independent experiments.


( C ) Clonogenic survival analysis and representative images of HeLa cells treated with the indicated siRNAs followed by I ‐ PpoI WT or I - PpoI H98A mRNA transfections . Survival ratio I - PpoI WT / I - PpoI H98A in each siRNA condition is presented . Error bars represent standard deviation and derive from 3 independent experiments .

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

(C) Clonogenic survival analysis and representative images of HeLa cells treated with the indicated siRNAs followed by I‐PpoI WT or I-PpoI H98A mRNA transfections. Survival ratio I-PpoI WT/I-PpoI H98A in each siRNA condition is presented. Error bars represent standard deviation and derive from 3 independent experiments.


( E ) 45S rDNA copy number analysis in patients ' tumors compared to adjacent controls in the LUAD cohort . 67 tumor samples and 43 samples from adjacent tissue were analyzed . ( F ) 45S rDNA copy number analysis in the LUAD patient samples based on RASSF1A CpG promoter methylation . 67 samples were analysed .

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

(E) 45S rDNA copy number analysis in patients' tumors compared to adjacent controls in the LUAD cohort. 67 tumor samples and 43 samples from adjacent tissue were analyzed. (F) 45S rDNA copy number analysis in the LUAD patient samples based on RASSF1A CpG promoter methylation. 67 samples were analysed.


F - M ) Total cellularity of F ) HSPCs , G ) LT - HSCs , H ) ST - HSCs , I ) Tom + HSCs , J ) GFP + drHSCs , K ) MPP2 , L ) MPP3 , M ) MPP4 in E16 . 5 fetuses following saline or maternal infection with Pru or RH as shown in Fig . 1A . n = 9 - 15 fetuses from at least 3 litters / condition .

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

F-M) Total cellularity of F) HSPCs, G) LT-HSCs, H) ST-HSCs, I) Tom+ HSCs, J) GFP+ drHSCs, K) MPP2, L) MPP3, M) MPP4 in E16.5 fetuses following saline or maternal infection with Pru or RH as shown in Fig. 1A. n = 9-15 fetuses from at least 3 litters/condition.


N - O ) Frequency of N ) HSCs ( CD150hi ) O ) MPPs ( CD150 - HSPCs ) expressing Ki67 ( G1 + G2 - M - S ) at E16 . 5 following saline or maternal infection with Pru or RH n = 9 - 15 fetuses from at least 3 litters / condition . For all analysis above bars represent mean + SEM . One - way ANOVA with Tukey ' s test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O

N-O) Frequency of N) HSCs (CD150hi) O) MPPs (CD150- HSPCs) expressing Ki67 (G1+G2-M-S) at E16.5 following saline or maternal infection with Pru or RH n = 9-15 fetuses from at least 3 litters/condition. For all analysis above bars represent mean + SEM. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


B ) Ratio of primary recipients with long - term multi - lineage reconstitution ( LTMR : > 1 % peripheral blood chimerism within each mature lineage ) in all 4 lineages ( granulocyte / macrophages [ GM ] , platelets , B - and T - cells ) , n is shown as the numerator . Statistical significance determined by Fisher ' s exact test ( 2 - tailed ) . * p ≤ 0 .

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 O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-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) Ratio of primary recipients with long-term multi-lineage reconstitution (LTMR: > 1% peripheral blood chimerism within each mature lineage) in all 4 lineages (granulocyte/macrophages [GM], platelets, B- and T-cells), n is shown as the numerator. Statistical significance determined by Fisher's exact test (2-tailed). *p ≤ 0.05


C - F ) Peripheral blood ( PB ) chimerism of primary Tom + HSC transplant recipient mice with LTMR in C ) granulocytes / macrophages ( GM ) , D ) platelets , E ) B - cells , and F ) T - cells at week 16 post - transplantation .

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

C-F) Peripheral blood (PB) chimerism of primary Tom+ HSC transplant recipient mice with LTMR in C) granulocytes/macrophages (GM), D) platelets, E) B-cells, and F) T-cells at week 16 post-transplantation.


G - J ) Peripheral blood ( PB ) chimerism of primary GFP + drHSC transplant recipient mice with LTMR in G ) granulocytes / macrophages ( GM ) , H ) platelets , I ) B - cells , and J ) T - cells at week 16 post - transplantation .

G-J) Peripheral blood (PB) chimerism of primary GFP+ drHSC transplant recipient mice with LTMR in G) granulocytes/macrophages (GM), H) platelets, I) B-cells, and J) T-cells at week 16 post-transplantation.


K ) Ratio of secondary recipients with long - term multi - lineage reconstitution ( LTMR ; > 0 . 1 % PB chimerism ) in all 4 lineages ( GM , platelets , B - and T - cells ) , n is shown as the numerator . Statistical significance determined by Fisher ' s exact test ( 2 - tailed ) . * p ≤ 0 .

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

K) Ratio of secondary recipients with long-term multi-lineage reconstitution (LTMR; > 0.1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells), n is shown as the numerator. Statistical significance determined by Fisher's exact test (2-tailed). *p ≤ 0.05


L - O ) Peripheral blood ( PB ) chimerism of secondary recipients of Tom + HSC with LTMR in L ) granulocytes / macrophages ( GM ) , M ) platelets , N ) B - cells , and O ) T - cells at week 16 post - transplantation .

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

L-O) Peripheral blood (PB) chimerism of secondary recipients of Tom+ HSC with LTMR in L) granulocytes/macrophages (GM), M) platelets, N) B-cells, and O) T-cells at week 16 post-transplantation.


P - S ) Peripheral blood ( PB ) chimerism of secondary recipients of GFP + drHSCs with LTMR in P ) granulocytes / macrophages ( GM ) , Q ) Platelets , R ) B - cells , and S ) T - cells at week 16 post - transplantation . No reconstitution ( nr ) was present in recipients of GFP + drHSCs in RH . Graphs C - J and L - S are plotted as the mean + SD . One - way ANOVA with Tukey ' s test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

P-S) Peripheral blood (PB) chimerism of secondary recipients of GFP+ drHSCs with LTMR in P) granulocytes/macrophages (GM), Q) Platelets, R) B-cells, and S) T-cells at week 16 post-transplantation. No reconstitution (nr) was present in recipients of GFP+ drHSCs in RH. Graphs C-J and L-S are plotted as the mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


A - I Measurement of A ) IFNγ , B ) IFNβ , C ) IL - 1β , D ) IL - 10 , E ) IL - 1α , F ) TNFα , G ) IL - 6 , H ) IL - 27 , and I ) GM - CSF cytokines in fetal amniotic fluid at E15 . 5 following prenatal infection . n = 8 - 9 fetuses from at least 3 litters / condition . For all analysis above bars represent mean + SD . One - way ANOVA with Tukey ' s test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE 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 O O O O O O O O O O O O O O O O O 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-I Measurement of A) IFNγ, B) IFNβ, C) IL-1β, D) IL-10, E) IL-1α, F) TNFα, G) IL-6, H) IL-27, and I) GM-CSF cytokines in fetal amniotic fluid at E15.5 following prenatal infection. n=8-9 fetuses from at least 3 litters/condition. For all analysis above bars represent mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; **p≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


U - C ' ) Measurement of U ) IFNγ , V ) IFNβ , W ) IL - 1β , X ) IL - 10 , Y ) IL - 1α , Z ) TNFα , A ' ) IL - 6 B ' ) IL - 27 , and C ' ) GM - CSF cytokines in maternal serum at E15 . 5 following prenatal infection ; n = 3 mice / condition . For all analysis above bars represent mean + SD . One - way ANOVA with Tukey ' s test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

U-C') Measurement of U) IFNγ, V) IFNβ, W) IL-1β, X) IL-10, Y) IL-1α, Z) TNFα, A') IL-6 B') IL-27, and C') GM-CSF cytokines in maternal serum at E15.5 following prenatal infection; n=3 mice/condition. For all analysis above bars represent mean + SD. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; **p≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


A - H ) Total cellularity of A ) HSPCs , B ) LT - HSCs , C ) Tom + HSCs , D ) GFP + drHSCs , E ) ST - HSCs , F ) MPP2 , G ) MPP3 , and H ) MPP4 at E15 . 5 following maternal IFNγ injection . For all analysis above , bars represent mean + SEM . n = 9 - 13 fetuses from 3 - 4 litters / condition . Statistical significance was determined by unpaired student ' s t - test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

A-H) Total cellularity of A) HSPCs, B) LT-HSCs, C) Tom+ HSCs, D) GFP+ drHSCs, E) ST-HSCs, F) MPP2, G) MPP3, and H) MPP4 at E15.5 following maternal IFNγ injection. For all analysis above, bars represent mean + SEM. n = 9-13 fetuses from 3-4 litters/condition. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


A ) Ratio of recipients with long - term multi - lineage reconstitution ( LTMR : > 1 % PB chimerism ) in all 4 lineages ( GM , platelets , B - and T - cells ) . N of mice is shown as the numerator .

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 O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O O

A) Ratio of recipients with long-term multi-lineage reconstitution (LTMR: > 1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells). N of mice is shown as the numerator.


B - E ) Peripheral blood ( PB ) chimerism of B ) granulocyte / macrophages ( GM ) , C ) platelets , D ) B - cells , and E ) T - cells in Tom + HSC transplant recipients at 16 weeks post - transplantation . F - I ) Peripheral blood ( PB ) chimerism of F ) granulocyte / macrophages ( GM ) , G ) platelets , H ) B - cells , and I ) T - cells in GFP + drHSC transplant recipients of mice at 16 weeks post - transplantation .

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

B-E) Peripheral blood (PB) chimerism of B) granulocyte/macrophages (GM), C) platelets, D) B-cells, and E) T-cells in Tom+ HSC transplant recipients at 16 weeks post-transplantation. F-I) Peripheral blood (PB) chimerism of F) granulocyte/macrophages (GM), G) platelets, H) B-cells, and I) T-cells in GFP+ drHSC transplant recipients of mice at 16 weeks post-transplantation.


J ) Ratio of secondary transplant recipients with long - term multi - lineage reconstitution ( LTMR > 0 . 1 % PB chimerism ) in all 4 lineages ( GM , platelets , B - and T - cells ) . N of mice is shown as the numerator .

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 O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O O

J) Ratio of secondary transplant recipients with long-term multi-lineage reconstitution (LTMR > 0.1% PB chimerism) in all 4 lineages (GM, platelets, B- and T-cells). N of mice is shown as the numerator.


K - N ) Peripheral blood ( PB ) chimerism of K ) granulocyte / macrophages ( GM ) , L ) platelets , M ) B - cells , and N ) T - cells in secondary Tom + HSC transplant recipients at 16 weeks post - transplantation . O - R ) Peripheral blood ( PB ) chimerism of Q ) granulocyte / macrophages ( GM ) , R ) Platelets , S ) B - cells , and T ) T - cells in secondary transplant recipients of GFP + drHSCs at 16 weeks post - transplantation . For all % chimerism experiments , mice not demonstrating LTMR were not included in analysis . Bars represent mean + SD . Statistical significance was determined by unpaired student ' s t - test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

K-N) Peripheral blood (PB) chimerism of K) granulocyte/macrophages (GM), L) platelets, M) B-cells, and N) T-cells in secondary Tom+ HSC transplant recipients at 16 weeks post-transplantation. O-R) Peripheral blood (PB) chimerism of Q) granulocyte/macrophages (GM), R) Platelets, S) B-cells, and T) T-cells in secondary transplant recipients of GFP+ drHSCs at 16 weeks post-transplantation. For all % chimerism experiments, mice not demonstrating LTMR were not included in analysis. Bars represent mean + SD. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01.


A ) IFNγ cytokine measured in the amniotic fluid ( AF ) and fetal liver supernatant ( FL ) of IFNγKO ( IFNγ - / - ) mice one day following injection of cytokine on E14 . 5 . ND = not detectable . Data represent mean of 3 fetuses / condition and error bars represent + SD .

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

A) IFNγ cytokine measured in the amniotic fluid (AF) and fetal liver supernatant (FL) of IFNγKO (IFNγ-/-) mice one day following injection of cytokine on E14.5. ND = not detectable. Data represent mean of 3 fetuses/condition and error bars represent + SD.


C - H ) Total cellularity of C ) HSPCs , D ) LT - HSCs , E ) ST - HSCs , F ) MPP2 , G ) MPP3 , and H ) MPP4 in saline or IFNγ exposed littermates of each genotype ( IFNγ + / - or - / - ) , as derived from the cross ( IFNγR + / - dam ) . n = 19 - 23 fetuses from 3 litters / condition .

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

C-H) Total cellularity of C) HSPCs, D) LT-HSCs, E) ST-HSCs, F) MPP2, G) MPP3, and H) MPP4 in saline or IFNγ exposed littermates of each genotype (IFNγ +/- or -/-), as derived from the cross (IFNγR +/- dam). n = 19-23 fetuses from 3 litters/condition.


J - O ) Total cellularity of J ) HSPCs , K ) LT - HSCs , L ) ST - HSCs , M ) MPP2 , N ) MPP3 , and O ) MPP4 in saline or IFNγ exposed littermates of each genotype ( IFNγ + / - or - / - ) , ( IFNγR - / - dam ) . n = 19 - 21 fetuses from 3 litters / condition . For all analysis bars represent mean . Statistical significance was determined by unpaired student ' s t - test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

J-O) Total cellularity of J) HSPCs, K) LT-HSCs, L) ST-HSCs, M) MPP2, N) MPP3, and O) MPP4 in saline or IFNγ exposed littermates of each genotype (IFNγ +/- or -/-), (IFNγR -/- dam). n = 19-21 fetuses from 3 litters/condition. For all analysis bars represent mean. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.


( A ) siRNA - treated BMDMs were infected for 16 hours with S . Typhimurium ( orgA - ) and LDH and IL - 1β release were measured .

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

(A) siRNA-treated BMDMs were infected for 16 hours with S. Typhimurium (orgA-) and LDH and IL-1β release were measured.


( B ) Cell death ( LDH ) and IL - 1β release evaluation in WT , Irgm2 - / - , GBPChr3 - / - and Casp11 - / - BMDMs infected for 16 hours with different Gram - negative bacteria ( MOI 25 ) .

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

(B) Cell death (LDH) and IL-1β release evaluation in WT, Irgm2-/-, GBPChr3-/- and Casp11-/- BMDMs infected for 16 hours with different Gram-negative bacteria (MOI 25).


( C ) Western blot examination of processed caspase - 1 ( p20 ) and gasdermin - D ( p30 ) in supernatants and pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) and GAPDH in cell lysates of WT and Irgm2 - / - BMDMs infected for 16 h with different Gram - negative bacterial strains .

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

(C) Western blot examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2-/- BMDMs infected for 16 h with different Gram-negative bacterial strains.


( D ) IL - 1β and cell death ( % LDH ) evaluation in immortalized WT , Irgm2 - / - , Casp11 - / - and Casp11 - / - Irgm2 - / - ( referred as sgIrgm2 ) BMDMs after 16 hours of E . coli , S . Typhimurium orgA - and OMV treatment .

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

(D) IL-1β and cell death (% LDH) evaluation in immortalized WT, Irgm2-/-, Casp11-/- and Casp11-/-Irgm2-/- (referred as sgIrgm2) BMDMs after 16 hours of E. coli, S. Typhimurium orgA- and OMV treatment.


( E ) Cell death ( % LDH ) evaluation in IFNγ - primed WT , Irgm2 - / - and Casp11 - / - BMDMs 4 hours after electroporation or not with 1µg of E . coli LPS .

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

(E) Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2-/- and Casp11-/- BMDMs 4 hours after electroporation or not with 1µg of E. coli LPS.


( F - H ) Survival of WT , Casp11 - / - and Irgm2 - / - mice primed with 100µg poly ( I : C ) for 6 hours and injected ( i . p . ) with 5mg / k - 1 LPS or 5 and 25 µg of OMVs ( n = 6 animals per condition ) .

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

(F-H) Survival of WT, Casp11-/- and Irgm2-/- mice primed with 100µg poly(I:C) for 6 hours and injected (i.p.) with 5mg/k-1 LPS or 5 and 25 µg of OMVs (n=6 animals per condition).


( A ) Measure of LDH and IL - 1β release in WT , GBPChr3 - / - and Casp11 - / - BMDMs were Irgm2 was knocked down 16 hours after exposure to 2 , 5 µg / 2 . 105 cells of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(A) Measure of LDH and IL-1β release in WT, GBPChr3-/- and Casp11-/- BMDMs were Irgm2 was knocked down 16 hours after exposure to 2,5 µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( B ) Cell death ( LDH ) and IL - 1β release evaluation in Irgm2 - silenced WT and GBPChr3 - / - BMDMs infected for 16 hours with either S . Tm orgA - or F . novicida ( MOI 25 ) . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(B) Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3-/- BMDMs infected for 16 hours with either S.Tm orgA- or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( C ) Florescence microscopy and associated quantifications of GBP - 2 ( green ) recruitments to intracellular S . Tm orgA - - mCherry ( MOI 10 , red ) in IFNγ - primed WT and Irgm2 - / - BMDMs . Nucleus was stained with Hoechst ( blue ) . Confocal images shown are from one experiment and are representative of n = 3 independent experiments ; scale bars 5 µm . For quantifications , the percentage of GBP - associated bacteria was quantified and ' n = ' refers to the number of intracellular bacteria counted in each experiment ; quantifications from n = 3 independent experiments were then plotted and expressed as mean ± SEM .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE 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 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) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM.


( D ) Confocal fluorescence microscopy images and associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red ) in IFNγ - primed iWT , iIrgm2 - / - , iGBPChr3 - / - and iGBPChr3 - / - / sgIrgm2 BMDMs after 8 hours of infection . Nucleus ( blue ) was stained with Hoescht ; scale bar 5µm . For quantifications , the percentage of bacteria positive for caspase - 11 - C254G - GFP was determined by combining the bacterial counts from n = 3 independent experiments and expressed as mean ± SEM . * * * p ≤ 0 . 001 for the indicated comparisons using t - test with bonferroni correction .

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

(D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.


( A ) GFP - Trap coupled to mass spectrometry strategy used . The volcano plot represents 3 independent combined experiments . Threshold selection of enriched proteins ( red dots ) in Irgm2 - GFP fraction was set at 2 fold enrichment ( x axis ) and p - value < 0 . 05 ( y axis ) . Blacks and Grey dots indicate proteins that did not reach a p - value < 0 . 05 using t - test with bonferroni correction . Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction .

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

(A) GFP-Trap coupled to mass spectrometry strategy used. The volcano plot represents 3 independent combined experiments. Threshold selection of enriched proteins (red dots) in Irgm2-GFP fraction was set at 2 fold enrichment (x axis) and p-value <0.05 (y axis). Blacks and Grey dots indicate proteins that did not reach a p-value <0.05 using t-test with bonferroni correction. Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction.


( B ) Green Florescent Protein ( GFP ) - Trap assay of the presence of Gate16 in Irgm2 - GFP - enriched fraction from the lysates of IFNγ - primed iIrgm2 - / - BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP ( Irgm2 - GFP ) or GFP alone . Arrows show the presence or not of Gate16 , Irgm2 - GFP and GAPDH in the Irgm2 - GFP - enriched fractions , flow - through and total cell lysates . * means non - specific

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

(B) Green Florescent Protein (GFP)-Trap assay of the presence of Gate16 in Irgm2-GFP-enriched fraction from the lysates of IFNγ-primed iIrgm2-/- BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP (Irgm2-GFP) or GFP alone. Arrows show the presence or not of Gate16, Irgm2-GFP and GAPDH in the Irgm2-GFP-enriched fractions, flow-through and total cell lysates. * means non-specific band


( C ) LDH and IL - 1β release from siRNA - treated WT BMDMs , and then exposed to 2 . 5 µg / 2 . 105 cells of OMVs for 16 hours .

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

(C) LDH and IL-1β release from siRNA-treated WT BMDMs, and then exposed to 2.5 µg/2.105 cells of OMVs for 16 hours.


( D ) LDH and IL - 1β release from WT , Irgm2 - / - and Casp11 - / - BMDMs silenced for Gate16 and treated for 16 hours with 2 , 5µg / 2 . 105 cells of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(D) LDH and IL-1β release from WT, Irgm2-/- and Casp11-/- BMDMs silenced for Gate16 and treated for 16 hours with 2,5µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( E ) Western blot examination of caspase - 11 and processed caspase - 1 ( p20 ) and gasdermin - D ( p30 ) in supernatants and pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) , pro - caspase - 11 , Gate16 and GAPDH in cell lysates of Gate16 - silenced WT , Irgm2 - / - and GBPChr3 - / - BMDMs exposed to 2 , 5 . 105µg / 2 . 105 cells of OMVs for 16 hours . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(E) Western blot examination of caspase-11 and processed caspase-1 (p20)and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55), pro-caspase-11, Gate16 and GAPDH in cell lysates of Gate16-silenced WT, Irgm2-/- and GBPChr3-/- BMDMs exposed to 2,5.105µg/2.105 cells of OMVs for 16 hours. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( F ) Representative confocal fluorescence microscopy images and associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red , MOI 10 ) in IFNγ - primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection . Nucleus ( blue ) was stained with Hoechst ; scale bar 5µm . For quantifications , the percentage of bacteria positive for caspase - 11 - C254G - GFP was determined by combining the bacterial counts from n = 3 independent experiments and expressed as mean ± SEM . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 for the indicated comparisons using t - test with bonferroni correction . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(F) Representative confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( A ) LDH and IL - 1B release from siRNA - treated primary human Monocyte - Derived Macrophages ( hMDMs ) infected with S . Typhimurium orgA - ( MOI25 ) for 16 hours . When specified , the caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or the NLRP3 inhibitor MCC950 ( 10µM ) were added to the experiments .

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

(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( B ) LDH and IL - 1B release from siRNA - treated primary human Monocyte - Derived Macrophages ( hMDMs ) , primed with IFNγ ( 10UI / mL ) and PAM3CSK4 ( 100ng / mL ) and then stimulated with Nigericin ( 20µM ) for 4 hours . When specified , the caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or the NLRP3 inhibitor MCC950 ( 10µM ) were added to the experiments .

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

(B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( C , D ) LDH and IL - 1B release from siRNA - treated WT or GBP1 / 2 / 5 - / - U937 Monocytic cell line , primed with IFNγ ( 10UI / mL ) and PAM3CSK4 ( 100ng / mL ) and then infected with ( C ) with S . Typhimurium orgA - ( MOI25 ) for 10 hours or ( D ) electroporated with 1µg of E . coli LPS for 4 hours . Φ indicates that no LPS electroporation was performed .

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

(C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed.


E ) H2AQ105me and H3K56ac levels in deletions of genes important for H3K56ac

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

E) H2AQ105me and H3K56ac levels in deletions of genes important for H3K56ac deposition


G - J ) ChIP - qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points . Samples were either normalized to input or to the core histone . n = 3 , biological replicates , error bars are standard error of the mean . * , p < 0 . 05 , relative to t = 0 , based on unpaired , 2 - tailed t -

O O O O B-EXP_ASSAY O B-EXP_ASSAY 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 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

G-J) ChIP-qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points. Samples were either normalized to input or to the core histone. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, relative to t=0, based on unpaired, 2-tailed t-test


B ) Volcano plot of quantitative SILAC experiments . Left upper quadrant indicates significantly , 2 - fold enriched proteins bound specifically to H2AQ105me . Members of the FACT complex ( SPT16 / POB3 ) are highlighted in red .

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B) Volcano plot of quantitative SILAC experiments. Left upper quadrant indicates significantly, 2-fold enriched proteins bound specifically to H2AQ105me. Members of the FACT complex (SPT16/POB3) are highlighted in red.


C ) Peptide pulldown using recombinantly purified Nhp2 . Eluted peptides were slot blotted and detected using streptavidine - HRP .

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C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were slot blotted and detected using streptavidine-HRP.


E ) ChIP - seq profile of Nhp2 ( orange ) and its respective input ( black ) across the entire chromosome XII of yeast . H2A - normalised H2AQ105me ( grey ) was directly taken as comparison . Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks . Scale bar is for the magnification .

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

E) ChIP-seq profile of Nhp2 (orange) and its respective input (black) across the entire chromosome XII of yeast. H2A-normalised H2AQ105me (grey) was directly taken as comparison. Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks. Scale bar is for the magnification.


D ) Proteomic analysis of Nhp2 interactors . Highlighted in red are the known members of the H / ACA pseudouridylation complex . The red dashed line indicates significance

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

D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoff


G ) Peptide pulldowns from human cells ( HEK293T ) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me . Dyskering ( Cbf5 homolog ) was probed to test for the presence of members of the H / ACA complex . Pulldown efficiency was analyzed by western blotting using the indicated antibodies .

O O O B-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

G) Peptide pulldowns from human cells (HEK293T) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me. Dyskering (Cbf5 homolog) was probed to test for the presence of members of the H/ACA complex. Pulldown efficiency was analyzed by western blotting using the indicated antibodies.


I ) Quantification of Northern Blot results . Normalised to GAPDH , n = 3 , error bars are standard error of the mean . Asterix indicates p - value ( * , p < 0 . 05 , based on unpaired , 2 - tailed t - test ) .

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

I) Quantification of Northern Blot results. Normalised to GAPDH, n=3, error bars are standard error of the mean. Asterix indicates p-value (*, p<0.05, based on unpaired, 2-tailed t-test).


A Hypocotyl lengths of wild - type and suc2 seedlings grown under continuous white light at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . The seedlings were grown on the medium with or without 3 % sucrose . Representative seedlings are shown in the upper panel . Different letters above each box indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n = 10 ) . Numbers indicate the ratio of hypocotyl lengths ( 28 ° C / 20 ° C ) . In the box plots ( A - D ) , the thick lines indicate median values , the lower and upper ends of the boxes represent the 25th and 75th percentiles , and the ends of the whiskers are set at 1 . 5 times the interquartile range .

O B-TISSUE B-EXP_ASSAY O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O 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 O O O O O O O O O O O B-TISSUE B-EXP_ASSAY 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 Hypocotyl lengths of wild-type and suc2 seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium with or without 3% sucrose. Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). In the box plots (A-D), the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


B Hypocotyl lengths of wild - type and tps1 - 2 ; GVG : : TPS1 seedlings grown under the same growth conditions Representative seedlings are shown in the upper panel . Different letters above each box indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n > 10 ) . Numbers indicate the ratio of hypocotyl lengths ( 28 ° C / 20 ° C ) .

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B Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under the same growth conditions Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C).


C Hypocotyl lengths of wild - type and tps1 - 2 ; GVG : : TPS1 seedlings grown under continuous white light at 20 ° C for 4 days followed by 3 days at 28 ° C . The seedlings were grown on the medium containing either mock or 20 µM of dexamethasone ( DEX ) . ns , not significant ( Student ' s t - test P ≥ 0 . 05 ; n > 10 ) . * * P < 0 . 01 ( Student ' s t - test ) ; n > 10 . Number indicates the ratio of hypocotyl lengths ( Dex / Mock ) .

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

C Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under continuous white light at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium containing either mock or 20 µM of dexamethasone (DEX). ns, not significant (Student's t-test P ≥ 0.05; n > 10). **P < 0.01 (Student's t-test); n > 10. Number indicates the ratio of hypocotyl lengths (Dex/Mock).


D Hypocotyl lengths of wild - type and tps1 - 2 ; GVG : : TPS1 seedlings grown in the dark at 20 ° C for 7 days . Representative seedlings are shown in the left panel . ns , not significant ( Student ' s t - test P ≥ 0 . 05 ; n > 10 ) .

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D Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown in the dark at 20 °C for 7 days. Representative seedlings are shown in the left panel. ns, not significant (Student's t-test P ≥ 0.05; n > 10).


F - I The qRT - PCR analyses of the transcript levels of YUC8 , IAA19 , IAA29 , and PIF4 . Transcript levels of each gene were normalized to those of PP2A and presented as values relative to those of wild - type seedlings at 20 ° C . Error bars indicate s . d . ( n = 3 ) . Different letters above each bar indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ) . Numbers indicate the ratio of transcript levels in seedlings grown at two different temperatures ( 28 ° C / 20 ° C ) .

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

F-I The qRT-PCR analyses of the transcript levels of YUC8, IAA19, IAA29, and PIF4. Transcript levels of each gene were normalized to those of PP2A and presented as values relative to those of wild-type seedlings at 20 °C. Error bars indicate s.d. (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). Numbers indicate the ratio of transcript levels in seedlings grown at two different temperatures (28 °C/20 °C).


A Western blotting with anti - PIF4 antibody shows that PIF4 protein levels are decreased in the tps1 - 2 ; GVG : : TPS1 mutants compared to wild - type plants . Seedlings were maintained under 12 - h light / 12 - h dark cycles at 20 ° C for 4 days and then transferred under continuous light on the 5th day . The growth temperature was increased to 28 ° C or kept at 20 ° C for 4 hours at ZT 20 - 24 before harvesting for total protein extraction . Equal loading of samples is shown by Ponceau S staining .

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

A Western blotting with anti-PIF4 antibody shows that PIF4 protein levels are decreased in the tps1-2;GVG::TPS1 mutants compared to wild-type plants. Seedlings were maintained under 12-h light/12-h dark cycles at 20 °C for 4 days and then transferred under continuous light on the 5th day. The growth temperature was increased to 28 °C or kept at 20°C for 4 hours at ZT 20-24 before harvesting for total protein extraction. Equal loading of samples is shown by Ponceau S staining.


B Western blotting showing the levels of PIF4 - Myc protein in PIF4 - OX and PIF4 - OX ; tps1 - 2 ; GVG : : TPS1 seedlings . Total protein was extracted from the seedlings grown under continuous white light at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Western blotting was probed using an anti - Myc antibody . Ponceau S staining is shown for equal loading .

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

B Western blotting showing the levels of PIF4-Myc protein in PIF4-OX and PIF4-OX;tps1-2;GVG::TPS1 seedlings. Total protein was extracted from the seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Western blotting was probed using an anti-Myc antibody. Ponceau S staining is shown for equal loading.


C , D Hypocotyl lengths in seedlings of different genotypes . Seedlings were grown under continuous white light at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( C ) . Numbers in ( D ) indicate the ratio of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n > 12 ) . In the box plots , the thick lines indicate median values , the lower and upper ends of the boxes represent the 25th and 75th percentiles , and the ends of the whiskers are set at 1 . 5 times the interquartile range .

O O O B-TISSUE B-EXP_ASSAY O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY 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 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 Hypocotyl lengths in seedlings of different genotypes. Seedlings were grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (C). Numbers in (D) indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 12). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


A , B Hypocotyl lengths of wild - type and KIN10 - OX seedlings grown under continuous white light at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( A ) . Numbers indicate the ratio of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n > 15 ) . In the box plots , the thick lines indicate median values , the lower and upper ends of the boxes represent the 25th and 75th percentiles , and the ends of the whiskers are set at 1 . 5 times the interquartile range .

O O O B-TISSUE B-EXP_ASSAY O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY 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 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 Hypocotyl lengths of wild-type and KIN10-OX seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (A). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


C Transient gene expression assays . The reporter vector containing 35S : : Renilla luciferase and IAA19p : : Firefly luciferase was co - transfected with 35S : : PIF4 and 35S : : KIN10 into Arabidopsis mesophyll protoplasts . The activity of Firefly luciferase under the control of IAA19 promoter was normalized to that of Renilla luciferase . Error bars indicate s . d . ( n = 4 ) . Different letters above each bar indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ) . T , 35S terminator .

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

C Transient gene expression assays. The reporter vector containing 35S::Renilla luciferase and IAA19p::Firefly luciferase was co-transfected with 35S::PIF4 and 35S::KIN10 into Arabidopsis mesophyll protoplasts. The activity of Firefly luciferase under the control of IAA19 promoter was normalized to that of Renilla luciferase. Error bars indicate s.d. (n = 4). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). T, 35S terminator.


D , E Hypocotyl lengths of seedlings grown under continuous white light at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( D ) . Numbers indicate the ratio of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n > 15 ) . In the box plots , the thick lines indicate median values , the lower and upper ends of the boxes represent the 25th and 75th percentiles , and the ends of the whiskers are set at 1 . 5 times the interquartile range .

O O O B-TISSUE B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY 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 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 Hypocotyl lengths of seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (D). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


B , C Yeast two - hybrid assays showing the direct interaction between PIF4 and KIN10 . Yeast clones were grown on the synthetic dropout medium with histidine ( + HIS ) or without histidine ( - HIS ) plus 25 mM of 3 - amino - 1 , 2 , 4 - triazole ( 3 - AT ) .

B, C Yeast two-hybrid assays showing the direct interaction between PIF4 and KIN10. Yeast clones were grown on the synthetic dropout medium with histidine (+HIS) or without histidine (-HIS) plus 25 mM of 3-amino-1,2,4-triazole (3-AT).


D Co - IP assays showing the interaction between PIF4 and KIN10 in vivo . Protein extracts from protoplasts expressing PIF4 - Myc and KIN10 - GFP ( or GFP ) were immunoprecipitated with anti - GFP antibody and analyzed by immunoblotting with anti - GFP or anti - Myc antibody .

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D Co-IP assays showing the interaction between PIF4 and KIN10 in vivo. Protein extracts from protoplasts expressing PIF4-Myc and KIN10-GFP (or GFP) were immunoprecipitated with anti-GFP antibody and analyzed by immunoblotting with anti-GFP or anti-Myc antibody.


E GRIK1 - activated KIN10 phosphorylates PIF4 . In vitro phosphorylation assays were performed at 30 ° C for 30 min using 1 μg of purified recombinant proteins ( KIN10 , GRIK1 , or PIF4 ) . 32P - labeled proteins were visualized by autoradiography . CBB , coomassie brilliant blue - stained gel .

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E GRIK1-activated KIN10 phosphorylates PIF4. In vitro phosphorylation assays were performed at 30 °C for 30 min using 1 μg of purified recombinant proteins (KIN10, GRIK1, or PIF4). 32P-labeled proteins were visualized by autoradiography. CBB, coomassie brilliant blue-stained gel.


F T6P inhibits PIF4 phosphorylation by KIN10 . This assay was performed with 1 μg of each protein at 30 ° C for 30 min in the presence of 1 mM T6P or sucrose ( Suc ) .

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F T6P inhibits PIF4 phosphorylation by KIN10. This assay was performed with 1 μg of each protein at 30 °C for 30 min in the presence of 1 mM T6P or sucrose (Suc).


The levels of PIF4 - Myc are negatively correlated with the levels of KIN10 - GFP . Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10 - GFP and PIF4 - Myc and analyzed by immunoblotting with anti - GFP or anti - Myc antibody . Ponceau S staining is shown for equal loading .

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The levels of PIF4-Myc are negatively correlated with the levels of KIN10-GFP. Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10-GFP and PIF4-Myc and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal loading.


KIN10 reduces the stability of PIF4 through 26S proteasome - mediated degradation . Transfected mesophyll protoplasts were pre - treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide ( CHX ) for the indicated time . Total protein was extracted from the protoplasts , and analyzed by immunoblotting with anti - GFP or anti - Myc antibody . Ponceau S staining is shown for equal protein loading . Representative western blots are shown in ( B ) .

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KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal protein loading. Representative western blots are shown in (B).


KIN10 reduces the stability of PIF4 through 26S proteasome - mediated degradation . Transfected mesophyll protoplasts were pre - treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide ( CHX ) for the indicated time . Total protein was extracted from the protoplasts , and analyzed by immunoblotting with anti - GFP or anti - Myc antibody . Quantification of relative PIF4 - Myc levels is shown in ( C ) . PIF4 - Myc levels are presented as values relative to those at 0 h . In ( C ) , different letters above each point indicate statistically significant differences ( ANOVA and Tukey ' s HSD ; P < 0 . 05 ; n = 3 ) . Error bars indicate s . d . ( n = 3 ) .

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KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3).


Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti - PIF4 antibody . Ponceau S staining shows equal protein loading .

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Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.


Western blotting showing the expression of RAP1 and TRF2 in MRC - 5 cells of different population doublings ( PD ) . Senescent cells ( PD 72 ) were left in culture for at least 4 weeks before harvesting for analysis .

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Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis.


ChIP analysis of young ( PD 30 ) and senescent ( PD 72 ) MRC - 5 cells performed with either anti - RAP1 , anti - TRF2 or an IgG antibody . The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe , stripped and hybridized to an Alu probe in order to determine unspecific binding . Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input . Data represent mean + / - SD of three biological replicates .

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ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2 or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean +/- SD of three biological replicates.


Immunofluorescence detection of 53BP1 ( red ) and a FISH probe staining telomeres ( green ) in young ( PD 26 ) , pre - senescent ( PD 66 ) and senescent ( PD 72 + 4 weeks ) MRC - 5 fibroblasts . The upper graph shows the percentage of telomere co - localizing with 53BP1 ( TIFs ) . Total DNA damage was measured by immunofluourescence detection of 53BP1 . The lower graph shows the total number of 53BP1 foci per nucleus . Approximately 40 - 50 cells were analyzed per replicate and per condition . Bars represent SEM of 2 biological replicates .

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Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66) and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluourescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40-50 cells were analyzed per replicate and per condition. Bars represent SEM of 2 biological replicates.


Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction ( 16p1 , 21q1 and XpYpM ) . The primer 16p1 is able to bind the subtelomeric region of 1p 9q , 12p , 15q , 16p , XqYq and the interstitial 2q14 region ; the 21q1 primer binds the subtelomeric region of 1q , 2q , 5q , 6q , 6p , 8p , 10q , 13q , 19p , 19q , 21q , 22q and the interstitial 2q13 site . The PCR - fusion assay was performed in MRC - 5 primary fibroblasts of different population doublings ( PD ) with or without depletion of RAP1 ( shRAP1 or shControl respectively ) . Lentiviral infection was performed for 10 days . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * * p < 0 . 001 ; * * * p < 0 . 0001 ) . Southern blotting showing the hybridization of the 16p probe for the conditions described in A . Number of fusions per 1 µg of DNA is shown at the bottom of the gels .

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

Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction (16p1, 21q1 and XpYpM). The primer 16p1 is able to bind the subtelomeric region of 1p 9q, 12p, 15q, 16p, XqYq and the interstitial 2q14 region; the 21q1 primer binds the subtelomeric region of 1q, 2q, 5q, 6q, 6p, 8p, 10q, 13q, 19p, 19q, 21q, 22q and the interstitial 2q13 site. The PCR-fusion assay was performed in MRC-5 primary fibroblasts of different population doublings (PD) with or without depletion of RAP1 (shRAP1 or shControl respectively). Lentiviral infection was performed for 10 days. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (**p < 0.001; ***p < 0.0001). Southern blotting showing the hybridization of the 16p probe for the conditions described in A. Number of fusions per 1 µg of DNA is shown at the bottom of the gels.


Number of fusions per 1 µg of DNA in MRC - 5 senescent cells . Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days ( two subsequent transfections of 3 days each ) . Representative southern blot membranes hybridized with the 16p probe are shown . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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Number of fusions per 1 µg of DNA in MRC-5 senescent cells. Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days (two subsequent transfections of 3 days each). Representative southern blot membranes hybridized with the 16p probe are shown. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).


Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1 . Variant telomere repeats were included in the estimation of the telomere array length . Data represent median + / - interquartile range ( green lines ) of 3 biological replicates . Statistical analysis was performed using Mann - Whitney U test * * * p < 0 . 0001 ) .

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Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1. Variant telomere repeats were included in the estimation of the telomere array length. Data represent median +/- interquartile range (green lines) of 3 biological replicates. Statistical analysis was performed using Mann-Whitney U test ***p < 0.0001).


RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days ( 20 µM final concentration ) . Fifteen days before cell harvesting , doxycycline ( DOX ; 1 µg / µl final concentration ) was added to deplete the expression of RAP1 .

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RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1.


Telomere length analysis by southern blotting of the samples described in A . The size of the main intensity peak is indicated at the bottom of the gel .

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Telomere length analysis by southern blotting of the samples described in A. The size of the main intensity peak is indicated at the bottom of the gel.


Number of fusions in HeLa cells after 25 days in culture . Cells were maintained with BIBR1532 during the whole period of the experiment , while doxycycline ( 1 µg / µl final concentration ) and the infection with shControl , shLIG3 or shLIG4 was carried out for the last 15 days of the experiment . Data represent mean + / - SD of three biological replicates are shown . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).


Examples of metaphase spreads hybridized with a telomeric PNA probe ( green ) when RAP1 was depleted ( + DOX ) . Telomerase was inhibited with BIBR1532 for 25 days . Scale bar = 10 µm . Quantification of chromosome aberrations observed in RAP1 depleted cells ( + DOX ) or control ( - DOX ) . Data represent mean + / - SD of three biological replicates . Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition . * p < 0 . 05 ; two - tailed Student ' s t test .

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Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm. Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.


Growth curve of MRC - 5 fibroblasts . Cells were grown until senescence and after three weeks of no growth detection , lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed . Cells were harvested 15 days post infection ( dpi ) . Data represent mean + / - SD of three biological replicates measured after 8 and 15 dpi .

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Growth curve of MRC-5 fibroblasts. Cells were grown until senescence and after three weeks of no growth detection, lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed. Cells were harvested 15 days post infection (dpi). Data represent mean +/- SD of three biological replicates measured after 8 and 15 dpi.


Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl , shp21CIP1 or shp21CIP1 + shRAP1 .

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Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1.


Dividing cells ( magenta ) were identified by EdU staining ( 1 µM EdU for 24 h ) . The percentage of dividing cells after 15 dpi is shown ( n = 3 ) . Scale bar = 10 µm .

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Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm.


Senescence - associated β - galactosidase ( SA - β - gal ) staining in senescent and post - senescent fibroblasts ( 1 and 15 dpi ) is shown . Percentage of SA - β - gal positive cells is indicated for each condition . Three biological replicates were performed , with approximately 300 cells analyzed per condition . Scale bar = 50 µm .

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Senescence-associated β-galactosidase (SA-β-gal) staining in senescent and post-senescent fibroblasts (1 and 15 dpi) is shown. Percentage of SA-β-gal positive cells is indicated for each condition. Three biological replicates were performed, with approximately 300 cells analyzed per condition. Scale bar = 50 µm.


Senescent cells , transduced with shp21CIP1 or shp21CIP1 + shRAP1 for 15 days , were stained with CytoCalcein violet ( live cells ) , Apopxin Green ( apoptotic cells ) and 7 - aminoactinomycin D ( 7 - AAD ) ( late apoptostic / necrotic cells ) and visualized by flow cytometry . Quantification of apoptotic cells from the conditions described in E . Data represent mean + / - SD of three biological replicates ( * * P < 0 . 001 ; two - tailed Student ' s t test ) .

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

Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry. Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).


A . Spike - binding antibodies in serum . Binding by antibodies of classes M , G , and A is detected with secondary antibodies conjugated to different fluorochromes , and the results are shown on the left , middle , and right plots , respectively . Binding ( geometric mean fluorescence intensity - GMFI - of corresponding secondary antibody ) to TE cells expressing SARS - CoV - 2 spike protein is plotted on the vertical axis , and binding to untransfected cells on the horizontal axis . Each point represents a value from one donor . Samples from donors with no known exposure to SARS - CoV - 2 antigens ( n = 86 ) are plotted with blue circles , recently infected donors ( n = 34 ) with red circles , and vaccinated donors ( n = 14 ) with green diamonds . Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated ( n = 5 , P values are derived from a two - way analysis of variance followed by Tukey ' s test to compare the specific binding , i . e . , ( binding to spike - expressing cells ) / ( binding to untransfected cells ) between conditions ( convalescent or vaccinated against unexposed ) within each antibody class . The experiment was independently repeated three times , and results shown come from the third replicate .

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

A. Spike-binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE cells expressing SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding to spike-expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.


B . Change in antibody binding following vaccination against SARS - CoV - 2 . Binding of antibodies from sera assessed in the experiment shown in ( A ) , is shown for two samples of serum from each of 7 donors . Data are plotted as in ( A ) with binding to spike - expressing cells on the vertical axis , and to untransfected cells on the horizontal , using blue symbols for samples from before vaccination , and red symbols for samples from 15 - 29 days after the first vaccination ( but before any second vaccination ) . Black lines connect points corresponding to pre / post pairs of samples from each donor . Left plot shows results for IgM and right plot for IgG . C . Comparison of post - vaccination increase in binding to untransfected cells between IgG and IgM . The increase in binding to untransfected TE 0 cells between pre - and post - vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM . The horizontal line at y = 0 is the expected result when no increased binding is observed . P value is derived from a two - tailed , Wilcoxon matched pairs signed rank test .

O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O B-TISSUE 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 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 O O O O O O O O O O O O O O O O O O O O O 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 O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-CELL_LINE 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 B-SUBCELLULAR 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

B. Change in antibody binding following vaccination against SARS-CoV-2. Binding of antibodies from sera assessed in the experiment shown in (A), is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike-expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15-29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG. C. Comparison of post-vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre- and post-vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y=0 is the expected result when no increased binding is observed. P value is derived from a two-tailed, Wilcoxon matched pairs signed rank test.


D . Comparison of spike - specific IgM , as measured by flow cytometry or ELISA . The left two " FC " are the results of flow cytometry , and the left verical axis shows the ratio of serum IgM binding to spike - expressing cells , vs binding to non - expressing control cells , as plottted in A . The right two columns show spike - RBD - specific IgM as measured by an IgM - capture ELISA , and the right vertical axis shows the optical densities ( OD ) . P values are calculated by two - way analysis of variance , followed by Sidak ' s multiple comparisons test . The entire experiment was repeated three times , and the data shown are derived from the third replicate .

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

D. Comparison of spike-specific IgM, as measured by flow cytometry or ELISA. The left two "FC" are the results of flow cytometry, and the left verical axis shows the ratio of serum IgM binding to spike-expressing cells, vs binding to non-expressing control cells, as plottted in A. The right two columns show spike-RBD-specific IgM as measured by an IgM-capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two-way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.