( E - I ) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide - induced Ulk1 puncta formation and Atg13 phosphorylation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) for the indicated times . ( E , F ) Cells were immunostained with an anti - Ulk1 antibody ( E ) , and the population of cells with Ulk1 puncta was calculated ( n > 100 cells in each experiment ) ( F ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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

(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (E, F) Cells were immunostained with an anti-Ulk1 antibody (E), and the population of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment) (F). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( E - I ) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide - induced Ulk1 puncta formation and Atg13 phosphorylation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) for the indicated times . ( G - I ) Cell lysates were collected in a time - dependent manner , and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting . Semiquantitative analyses are shown in Fig . EV3D .

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

(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (G-I) Cell lysates were collected in a time-dependent manner, and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting. Semiquantitative analyses are shown in Fig. EV3D.


( J ) EGFP - DFCP1 - expressing PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for the indicated times , followed by immunostaining with an anti - Ulk1 antibody . Representative images of EGFP - DFCP1 ( green ; left ) , anti - Ulk1 ( red ; center ) , and a merged image ( right ) are shown . The nuclei were stained with Hoechst 33342 in the merged image . A magnified image of the area within the dashed square is also shown . Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE 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 B-GENEPROD O B-GENEPROD 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-SUBCELLULAR 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 B-GENEPROD O O O O B-GENEPROD O O

(J) EGFP-DFCP1-expressing PPM1D+/+MEFs were treated with etoposide (10 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images of EGFP-DFCP1 (green; left), anti-Ulk1 (red; center), and a merged image (right) are shown. The nuclei were stained with Hoechst 33342 in the merged image. A magnified image of the area within the dashed square is also shown. Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta.


( K , L ) The role of Ulk1 dephosphorylation at Ser637 on etoposide - induced DFCP1 puncta formation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were transfected with EGFP - DFCP1 and treated with etoposide ( 10 µM ) for the indicated hours , followed by immunostaining with an anti - HA antibody . Representative merged images of EGFP - DFCP1 ( green ) , anti - HA ( red ) , and Hoechst 33342 ( blue ) are shown in ( K ) . Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta . The number of DFCP1 puncta in each cell was calculated and is shown in ( L ) ( n > 8 cells in each experiment ) . Red lines indicate the mean values . * * p < 0 . 01 , NS : not significant .

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

(K, L) The role of Ulk1 dephosphorylation at Ser637 on etoposide-induced DFCP1 puncta formation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were transfected with EGFP-DFCP1 and treated with etoposide (10 µM) for the indicated hours, followed by immunostaining with an anti-HA antibody. Representative merged images of EGFP-DFCP1 (green), anti-HA (red), and Hoechst 33342 (blue) are shown in (K). Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. The number of DFCP1 puncta in each cell was calculated and is shown in (L) (n > 8 cells in each experiment). Red lines indicate the mean values. **p < 0.01, NS: not significant.


( A ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 , induction of autophagy , and degradation of Noxa . PPM1D + / + and PPM1D − / − primary thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , thymocytes were lysed and the expression of each protein was examined by western blotting . α - Tubulin was used as a loading control . Semiquantitative analyses are shown in Fig . EV5B .

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

(A) PPM1D-dependent dephosphorylation of Ulk1 at Ser637, induction of autophagy, and degradation of Noxa. PPM1D+/+ and PPM1D−/− primary thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were lysed and the expression of each protein was examined by western blotting. α-Tubulin was used as a loading control. Semiquantitative analyses are shown in Fig. EV5B.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( B , C ) The indicated thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , thymocytes were immunostained with an anti - LC3 antibody . Nuclei were stained with Hoechst 33342 . Representative images are shown in ( B ) . LC3 puncta can be seen in irradiatedPPM1D + / + thymocytes ( arrowheads ) . In ( C ) , the proportion of cells with LC3 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 .

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

(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (B, C) The indicated thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were immunostained with an anti-LC3 antibody. Nuclei were stained with Hoechst 33342. Representative images are shown in (B). LC3 puncta can be seen in irradiatedPPM1D+/+thymocytes (arrowheads). In (C), the proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( D ) The indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , thymocytes were subjected to EM analysis . An autophagic vacuole ( arrowhead ) can be seen in an irradiated PPM1D + / + thymocyte .

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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+thymocyte.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( E ) The indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , thymocytes were stained with Cyto - IDTM . Representative flow cytometry data are shown . Quantitative data are shown in Fig . EV5C . Cyto - ID fluorescence in irradiated PPM1D + / + thymocytes was higher than that in the other thymocytes .

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

(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (E) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were stained with Cyto-IDTM. Representative flow cytometry data are shown. Quantitative data are shown in Fig. EV5C. Cyto-ID fluorescence in irradiated PPM1D+/+thymocytes was higher than that in the other thymocytes.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( F ) Analysis of autophagic flux . The indicated thymocytes were X - rayirradiated ( 5 Gy ) in the presence or absence of chloroquine ( 120 µM ) , and the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . Semiquantitative analyses are shown in Figure EV4D .

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

(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (F) Analysis of autophagic flux. The indicated thymocytes were X-rayirradiated (5 Gy) in the presence or absence of chloroquine (120 µM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. Semiquantitative analyses are shown in Figure EV4D.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( G ) Thymocytes were X - ray irradiated ( 5 Gy ) , and 6 hr later , cell death was determined using the PI assay . Data represent the mean ± SD ( n = 3 independent thymi ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

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(G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (G) Thymocytes were X-ray irradiated (5 Gy), and 6 hr later, cell death was determined using the PI assay. Data represent the mean  ± SD (n = 3 independent thymi). *p < 0.05, **p < 0.01, NS: not significant.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( H , I ) Thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . ( I ) Semiquantitative analyses are shown ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 .

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

(G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (H, I) Thymocytes were X-rayirradiated (5 Gy), and 3 hr later, the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses are shown (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( J ) Indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , caspase3 / 7 activity was examined using the Caspase - Glo 3 / 7 assay ( Promega ) according to the manufacturer ' s protocol . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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

(G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (J) Indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, caspase3/7 activity was examined using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's protocol. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( K ) The effect of Noxa siRNA on thymocytes . The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs . After 12 hr , cells were X - ray irradiated ( 5 Gy ) , and 6 hr or 10 hr later , the population of dead cells among the GFP - positive cells was determined using the PI assay . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , NS : not significant .

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

(K) The effect of Noxa siRNA on thymocytes. The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs. After 12 hr, cells were X-ray irradiated (5 Gy), and 6 hr or 10 hr later, the population of dead cells among the GFP-positive cells was determined using the PI assay. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, NS: not significant.


( F ) Single tube qPCR experiments showing the down - regulation of the synaptic genes NLGN3 , SLC6A , TRIM9 , SYT1 , SYNGR1 , and SNAP91 in ALSC9orf72 MN ( Welch ' s t - test ) . n = 3 independent cultures for each hiPSC line . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(F) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( G - H ) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures ( two - way ANOVA ) . n = 3 independent cultures for each hiPSC line ( for each time point ) . Scale bar : 25 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures (two-way ANOVA). n=3 independent cultures for each hiPSC line (for each time point). Scale bar: 25 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( A ) Both K + channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with each hiPSC line . Scale bar : 50 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-DISEASE O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Both K+ channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 50 µm Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( C ) Apamin and XE991 decrease the size of aberrant aggresomes , confirming enhanced autophagy degradation ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with the ALSC9orf72 I line . Scale bar : 5 Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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(C) Apamin and XE991 decrease the size of aberrant aggresomes, confirming enhanced autophagy degradation (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with the ALSC9orf72 I line. Scale bar: 5 Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) The load of aggregated SQSTM1 / p62 is also reduced by the treatments in ALSC9orf72 cultures ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments for each cell line . Scale bar : 20 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(E) The load of aggregated SQSTM1/p62 is also reduced by the treatments in ALSC9orf72 cultures (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments for each cell line. Scale bar: 20 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( F ) Treatment with the K + channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN . n = 3 independent treatments with each hiPSC line ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . Scale bar : 5 µm . Dashed line represents the cell soma . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Treatment with the K+ channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN. n=3 independent treatments with each hiPSC line (one-way ANOVA followed by Dunnett's multiple comparison test). Scale bar: 5 µm. Dashed line represents the cell soma. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with each hiPSC line . Scale bar : 10 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent SEM . Exact p - values are reported in Appendix Table S1 .

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

(E) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( F ) ALSC9orf72 MN showed an increased number of Homer1b / c + synaptic contacts after treatment with the K + channel blockers , which in contrast had no significant effect on Healthy MN . n = 3 independent treatments with each hiPSC line ( two - way ANOVA ) . Scale bar : 5 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent SEM . Exact p - values are reported in Appendix Table S1 .

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(F) ALSC9orf72 MN showed an increased number of Homer1b/c+ synaptic contacts after treatment with the K+ channel blockers, which in contrast had no significant effect on Healthy MN. n=3 independent treatments with each hiPSC line (two-way ANOVA). Scale bar: 5 µm Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( K ) Synapse - and neuronal processes - related proteins significantly up - regulated by both treatments .

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(K) Synapse- and neuronal processes-related proteins significantly up-regulated by both treatments.


( A - B ) Levels of miR - 122 in Huh7 cells and released EVs either untreated ( Fed ) or subjected to starvation for metabolites including amino acids for 16h ( Starved ) . miR - 122 signals were detected by Northern blotting and position of the 32P - labelled Oligos that served as size markers are shown in the lane ( A ) . U6 snRNA was used as loading control .

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(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control.


( A - B ) Levels of miR - 122 in Huh7 cells and released EVs either untreated ( Fed ) or subjected to starvation for metabolites including amino acids for 16h ( Starved ) . Cellular CAT - 1 levels were measured by qRT - PCR using GAPDH mRNA as control ( B ) . A scheme of experiment is shown in the top panel in B .

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(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.


( C - D ) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells . Relative changes in cellular and extracellular levels ( EV associated ) of miR - 122 and miR - 24 in Fed or 8h Starved Huh7 cells were quantified by qRT - PCR and plotted ( mean + / - s . e . m . , n = 3 ) . Levels of individual miRNAs in Fed condition were taken as unit . Fold change ( with SD ) in the cellular level of all miRNAs and pre - miR - 122 measured were shown in the bottom panel ( C ) . Cellular miRNA levels were normalized against U6 snRNA .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV associated) of miR-122 and miR-24 in Fed or 8h Starved Huh7 cells were quantified by qRT-PCR and plotted (mean+/- s.e.m., n=3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of all miRNAs and pre-miR-122 measured were shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA.


( C - D ) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells . Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions . CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels . Mean of three independent measurements are plotted ( middle panel in D ) . Calnexin and GAPDH are not detected in EVs but visible in the cellular extract . EV associated HA - HuR levels for Fed and Starved Huh7 cells expressing HA - HuR were also detected in the bottom panel in D .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements are plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV associated HA-HuR levels for Fed and Starved Huh7 cells expressing HA-HuR were also detected in the bottom panel in D.


( E ) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells . Levels of miRNAs were measured by real - time quantification and normalized against U6 snRNA . Mean data are from three independent experiments . DMSO treatment was used as control for GW4869 treated cells .

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(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.


( F ) CAT - 1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells . qRT - PCR techniques was adopted for quantification using 18S rRNA values for normalization ( mean + / - s . e . m . , n = 3 ) .

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(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).


( G ) Effect of siSMPD2 treatment on miR - 122 content of EVs from Fed and Starved Huh7 cells . EVs from Fed and Starved Huh7 cells , depleted for SMPD2 , were isolated and miR - 122 content were measured by qRT - PCR and normalized against protein content of EVs . Fold change in EV associated miR - 122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars .

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(G) Effect of siSMPD2 treatment on miR-122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 content were measured by qRT-PCR and normalized against protein content of EVs. Fold change in EV associated miR-122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars.


( H ) Effect of siSMPD2 treatment on CAT - 1 mRNA content ( left panel ) and miR - 122 levels ( right panel ) in Huh7 cells . RNAs , from Fed and Starved Huh7 cells , depleted for SMPD2 , were isolated and miR - 122 and CAT - 1 mRNA contents were measured by qRT - PCR . miR - 122 contents were normalized against U6 snRNA . GAPDH mRNA was used for normalization of CAT - 1 mRNA levels ( mean + / - s . e . m . , n = 3 ) . miR - 122 levels for siCon treated cells ( Fed and Starved ) were considered as units .

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(H) Effect of siSMPD2 treatment on CAT-1 mRNA content (left panel) and miR-122 levels (right panel) in Huh7 cells. RNAs, from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 and CAT-1 mRNA contents were measured by qRT-PCR. miR-122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT-1 mRNA levels (mean +/- s.e.m., n=3). miR-122 levels for siCon treated cells (Fed and Starved) were considered as units.


( I - J ) A schematic representation of the experiment to separate Extracellular Vesicles ( EVs ) on OptiPrep @ density gradient ( left panel ) . Densities of fractions 1 - 10 are plotted and a best - fit curve is drawn ( right upper panel ) . CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells ( right lower panel ) . Mean Ct values of miR - 122 in RNAs isolated from Exosome enriched ( 8 - 9 ) vs Non - exosomal ( 1 - 3 ) fractions were analyzed and plotted ( J ) ( mean + / - s . e . m . , n = 3 ) .

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(I-J) A schematic representation of the experiment to separate Extracellular Vesicles (EVs) on OptiPrep@ density gradient (left panel). Densities of fractions 1-10 are plotted and a best-fit curve is drawn (right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells (right lower panel). Mean Ct values of miR-122 in RNAs isolated from Exosome enriched (8-9) vs Non-exosomal (1-3) fractions were analyzed and plotted (J) (mean +/- s.e.m., n=3).


( K ) Effect of Starvation on apoptic cell numbers in Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells . β - Actin and CD63 are used as loading control for cellular and EV associated fraction respectively ( left panel ) . TUNEL positive cells in Fed and Starved Huh7 cells . TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified . Fed , but DNase treated , cells were used as positive control ( + ve control ) ( right panel ) .

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(K) Effect of Starvation on apoptic cell numbers in Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells. β-Actin and CD63 are used as loading control for cellular and EV associated fraction respectively (left panel). TUNEL positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified. Fed, but DNase treated, cells were used as positive control (+ve control) (right panel).


( L ) Effect of thapsigargain ( TG ) treatment of Huh7 cells on cellular and EV associated miR - 122 level . A schematic representation of the experiment ( upper panel ) . miR - 122 levels were measured by qRT - PCR in total cellular RNA and in EVs released by the treated cells . Values were normalized either against U6 snRNA or protein content of EVs ( mean + / - s . e . m . , n = 3 ) ( lower panels ) for cellular and EV associated RNA respectively .

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(L) Effect of thapsigargain (TG) treatment of Huh7 cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.


( M ) Effect of TG on cellular level of eIF2 - a and its phosphorylated form , and Ago2 and CD63 in EVs . b - Actin was used as loading control for cellular samples .

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(M) Effect of TG on cellular level of eIF2-a and its phosphorylated form, and Ago2 and CD63 in EVs. b-Actin was used as loading control for cellular samples.


( A ) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells . Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR - 122 and miR - 21 were done by quantitative RT - PCR ( mean + / - s . e . m . , n = 3 ) . GFP used as negative control to confer the specificity of the anti - HuR3A2 antibody in immunoprecipitating HuR . The miR - 122 content in the GFP - antibody immunoprecipitated materials was too low to get detected reliably using the qRT - PCR .

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(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR.


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Schemes of HuR depletion experiments in Huh7 cells ( B ) . In siHuR treated cells , depletion of HuR was monitored by western blotting ( mean + / - s . e . m . , n = 4 )

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4)


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT - PCR . Cellular miRNA contents were normalized against U6 snRNA . Levels of each miRNA in control siRNA ( siCon ) treated set were taken as unit . ( C ) . CAT - 1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT - PCR and normalized against GAPDH mRNA .

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA.


( D ) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells . Relative levels were estimated by real time qPCR , normalized with respect to U6 snRNA and plotted in the left panel . Levels of miR - 122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT - PCR and plotted in the right panel ( mean + / - s . e . m . , n = 3 ) .

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(D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3).


( E ) Level of eIF - 2a and its phosphorylated form , estimated by western blot using specific antibodies , in siCon or siHuR treated and starved Huh7 cell extracts . Western blot data of HuR confirm reduction of this protein upon siRNA treatment . CAT - 1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level . Values of siCon treated samples were taken as unit ( mean + / - s . e . m . , n = 3 ) . In the right panel the relative quantity of phosphorylated eIF - 2a measured by densitometry were plotted . Levels of p38 and phosphor - p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot .

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(E) Level of eIF-2a and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2a measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot.


( F ) Western blot analysis to detect the level of phosphorylated eIF - 2a and eIF - 4E - BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant - let - 7a or anti - miR - 122 oligos . miR - 122 inactivation was monitored by measuring cat - 1 level by qRT - PCR using GAPDH as control ( mean + / - s . e . m . , n = 3 ) . * denotes the phosphorylated eIF - 4E - BP1 . b - Actin was used as loading control . Relative levels of phosphorylated eIF - 2a were measured by densitometric estimation of three independent measurements .

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(F) Western blot analysis to detect the level of phosphorylated eIF-2a and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. b-Actin was used as loading control. Relative levels of phosphorylated eIF-2a were measured by densitometric estimation of three independent measurements.


( A ) Northern blot of miR - 122 in Huh7 cells expressing the Control and HA - HuR expression plasmids . The U6 was used for loading control . Position of the 32P - labelled Oligos , blotted to the membrane used for Northern blotting , served as size markers are shown in the M lane .

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(A) Northern blot of miR-122 in Huh7 cells expressing the Control and HA-HuR expression plasmids. The U6 was used for loading control. Position of the 32P-labelled Oligos, blotted to the membrane used for Northern blotting, served as size markers are shown in the M lane.


( B ) Schematic representation of experiments done in Huh7 cells expressing HA - HuR ( bottom ) . For control , pCIneo transfected Huh7 cells were used . Relative expression of HuR in transfected cells were analysed by western blot ( top ) . b - actin blots were used as loading controls .

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(B) Schematic representation of experiments done in Huh7 cells expressing HA-HuR (bottom). For control, pCIneo transfected Huh7 cells were used. Relative expression of HuR in transfected cells were analysed by western blot (top). b-actin blots were used as loading controls.


( C ) Cellular and EV - associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA - HuR expressing vectors by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . Cellular miRNA levels were normalized against U6 snRNA .

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(C) Cellular and EV-associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA-HuR expressing vectors by qRT-PCR (mean+/- s.e.m., n=5). Cellular miRNA levels were normalized against U6 snRNA.


( E ) Effect of HA - HuR expression on miRNA levels on EV and non - EV fractions of the culture supernatant collected from control and HA - HuR encoding plasmid transfected Huh7 cells . For isolation of non - EV fraction , supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation ( left panel ) ( mean + / - s . e . m . , n = 3 ) . Effect of HuR expression on CD63 , Alix and HuR levels in EVs . Extracts from EVs isolated from control or HA - HuR expressing Huh7 cells were western blotted for respective proteins ( right panel ) .

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

(E) Effect of HA-HuR expression on miRNA levels on EV and non-EV fractions of the culture supernatant collected from control and HA-HuR encoding plasmid transfected Huh7 cells. For isolation of non-EV fraction, supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation (left panel) (mean+/- s.e.m., n=3). Effect of HuR expression on CD63, Alix and HuR levels in EVs. Extracts from EVs isolated from control or HA-HuR expressing Huh7 cells were western blotted for respective proteins (right panel).


( F ) Relative miR - 122 levels in CD63 positive affinity purified EVs isolated from HA - HuR and pCIneo transfected Huh7 cells were quantified by qRT - PCR ( mean + / - s . e . m . , n = 3 ) . The levels of miR - 122 were normalized against CD63 content of EVs .

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(F) Relative miR-122 levels in CD63 positive affinity purified EVs isolated from HA-HuR and pCIneo transfected Huh7 cells were quantified by qRT-PCR (mean+/- s.e.m., n=3). The levels of miR-122 were normalized against CD63 content of EVs.


( G ) Effect of GW4869 on EV associated content of three different miRNAs in HA - HuR expressing Huh7 cells quantified by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . EVs from DMSO treated cells were used as control .

O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE 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 B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O

(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.


( H ) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA - HuR or pCIneo respectively by running OptiPrepTM density gradient ( left panel ) and western blotting for CD63 ( right upper panel ) . Relative miR - 122 levels in fractions 1 - 2 and 8 - 9 were compared by qRT - PCR ( right lower panel ) .

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(H) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA-HuR or pCIneo respectively by running OptiPrepTM density gradient (left panel) and western blotting for CD63 (right upper panel). Relative miR-122 levels in fractions 1-2 and 8-9 were compared by qRT-PCR (right lower panel).


( I ) Levels of apoptic cells in HA - HuR expressing Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C was done ( top ) for EV associated and cellular fractions of HA - HuR and pCIneo ( control ) expressing Huh 7 cells respectively . Western blot for HA - HuR was done to analyse the expression of HA - HuR in EVs isolated from HA - HuR and pCIneo expressing Huh7 cells . CD63 and β - actin are used as loading control for EV associated fractions and cellular fractions respectively ( upper panels ) . TUNEL assay of HA - HuR and pCIneo expressing and non - transfected Huh 7 cells was performed and representative pictures of HA - HuR and pCIneo expressing cells along with picture of non - transfected but DNase treated ( positive control ) cells were compared ( lower panels ) . Scale bar 50 µM .

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

(I) Levels of apoptic cells in HA-HuR expressing Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C was done (top) for EV associated and cellular fractions of HA-HuR and pCIneo (control) expressing Huh 7 cells respectively. Western blot for HA-HuR was done to analyse the expression of HA-HuR in EVs isolated from HA-HuR and pCIneo expressing Huh7 cells. CD63 and β-actin are used as loading control for EV associated fractions and cellular fractions respectively (upper panels). TUNEL assay of HA-HuR and pCIneo expressing and non-transfected Huh 7 cells was performed and representative pictures of HA-HuR and pCIneo expressing cells along with picture of non-transfected but DNase treated (positive control ) cells were compared (lower panels). Scale bar 50 µM.


( B ) Change in miR - 122 level in starved mouse liver . Endogenous miR - 122 ( left panel ) and CAT - 1 mRNA ( middle panel ) levels in mouse liver starved for 12h were quantified by qRT - PCR . miR - 122 levels in the blood serum of corresponding animals were also measured ( right panel ) . The Ct values of miR - 122 and CAT - 1 mRNA obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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

(B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( C ) Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots , β - actin was used as loading control .

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(C)Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control .


( E ) Western Blot analysis to check expression of HA - HuR in liver samples of injected mice . β - Actin was used as loading control . HA - HuR expressing Huh7 cells lysate was used as positive control .

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(E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control.


( F ) . Change in miR - 122 level upon expression of HA - HuR in mouse liver . Endogenous miR - 122 level in HA - HuR expression plasmid injected mouse liver were quantified by RT - PCR and compared against liver RNA samples collected from control ( pCIneo injected ) group of animals ( mean + / - s . e . m . , n = 5 ) .

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(F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5).


( G ) Endogenous miR - 122 target CAT - 1 ( left panel ) and Aldolase ( right panel ) mRNA levels in HA - HuR or pCIneo ( control ) injected mouse liver were quantified by RT - PCR ( mean + / - s . e . m . , n = 4 ) .

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(G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4).


( H ) miR - 122 levels in the blood serum of pCIneo ( control ) or HA - HuR expression plasmid injected mice were measured . The Ct values of miR - 122 and CAT - 1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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

(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( A ) Time course experiment of HuR binding of miR - 122 replaced from Ago2miRNPs . A schematic representation of in vitro miRNA binding assay of HuR ( left panel ) . Equal amounts of recombinant HuR ( rHuR ) , after indicated time of the miRNP interaction on FLAG - beads , were immunoprecipitated and HuR associated miR - 122 levels were detected by qRT - PCR ( right top panel ) . Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials . HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials , at different time points ( right bottom panel ) .

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

(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).


( C ) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay . The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation . The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti - HuR antibody .

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(C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody.


( D ) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials . A representative domain picture of HuR and HuRΔIII are shown in the upper panel . Relative levels of HuR bound miR - 122 were determined by qRT - PCR ( mean + / - s . e . m . , n = 3 ) ( middle left panel ) . Amount of miR - 122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials . Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials ( bottom panel ) .

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

(D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel).


( E ) RNA electrophoretic mobility shift assay done with 32P - labeled synthetic miR - 122 and recombinant HuR proteins . Radiolabelled miR - 122 ( 100 fmol ) was incubated with increasing concentration of FL - HuR ( 5 - 100nM ) or HuRΔIII ( 100 nM ) and gel shifting of miR - 122 were marked by arrowhead . Position of the free probes is marked by an arrow . In the right panel , 1pmol of unlabelled TNF - alpha AU - rich element encoding synthetic RNA was used to compete with miR - 122 binding of full length HuR . Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page .

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

(E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page.


( F ) Association of miRNAs with recombinant HuR in vitro . Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA , recovered from immunoprecipitated HuR after the binding reaction , were quantified along with the input RNA used for binding . The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted ( mean + / - s . e . m . , n = 3 ) . The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot .

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(F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.


( A ) Subcellular distribution of HuR and Ago2 in Huh7 cells . Isotonic lysates of Huh7 cells transfected with HA - HuR expression or pCIneo control vector were analyzed on 3 - 30 % OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis . Alix and Calnexin were used as markers of late endosome / MVB and ER respectively . Positions of specific organelle containing fractions were marked above the panels . * denotes the position of the high molecular weight HuR bands detected in the MVB fractions .

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

(A) Subcellular distribution of HuR and Ago2 in Huh7 cells. Isotonic lysates of Huh7 cells transfected with HA-HuR expression or pCIneo control vector were analyzed on 3-30% OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis. Alix and Calnexin were used as markers of late endosome/MVB and ER respectively. Positions of specific organelle containing fractions were marked above the panels. * denotes the position of the high molecular weight HuR bands detected in the MVB fractions.


( B ) Detection of ubiquitinated HuR in Huh7 cells expressing HA - Ub . HuR was immunoprecipitated with anti - HuR antibody from cell lysate of Huh7 cells transfected with HA - Ub expression or pCIneo ( control ) plasmids and western blotted for HA or HuR ( left panel ) . In vitro ubiquitination assay was done with recombinant His - tagged HuR and cell extract of Huh7 cells expressing HA - Ub . The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown . Specific bands of Ub - HuR are marked by arrows and HuR is marked by arrowhead .

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

(B) Detection of ubiquitinated HuR in Huh7 cells expressing HA-Ub. HuR was immunoprecipitated with anti-HuR antibody from cell lysate of Huh7 cells transfected with HA-Ub expression or pCIneo (control) plasmids and western blotted for HA or HuR (left panel). In vitro ubiquitination assay was done with recombinant His-tagged HuR and cell extract of Huh7 cells expressing HA-Ub. The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown. Specific bands of Ub-HuR are marked by arrows and HuR is marked by arrowhead.


( C ) Changes in relative level of miR - 122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA - HuR expression vector transfected cells were measured by qRT - PCR ( mean + / - s . e . m . , n = 5 ) .

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(C) Changes in relative level of miR-122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA-HuR expression vector transfected cells were measured by qRT-PCR (mean+/- s.e.m., n=5).


( D ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells . Organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR . Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins . Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads . Levels of HuR in the immunoprecipitated materials were detected using anti - HuR antibody . Anti - GFP antibody immnoprecipitated materials were used as control .

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(D) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells. Organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR. Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins. Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads. Levels of HuR in the immunoprecipitated materials were detected using anti-HuR antibody. Anti-GFP antibody immnoprecipitated materials were used as control.


( E ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co - expressing HA - Ubiquitin and FLAG - HuR . Cells were lysed and organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR . HuR in the lysate of the FLAG - HuR and HA - Ub co expressing cells were detected by HuR specific antibody .

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

(E) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co-expressing HA-Ubiquitin and FLAG-HuR. Cells were lysed and organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR. HuR in the lysate of the FLAG-HuR and HA-Ub co expressing cells were detected by HuR specific antibody.


( F ) Loss of miRNA association of HuR on MVB . HA - HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA - HuR and associated miRNA levels were measured by qRT - PCR and normalized against immunoprecipitated proteins . Levels of immunoprecipitated HA - HuR were detected by western blotting and quantified by densitometry . Data represented as mean + / - s . e . m . , n = 3 .

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

(F) Loss of miRNA association of HuR on MVB. HA-HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA-HuR and associated miRNA levels were measured by qRT-PCR and normalized against immunoprecipitated proteins. Levels of immunoprecipitated HA-HuR were detected by western blotting and quantified by densitometry. Data represented as mean+/- s.e.m., n=3.


( G ) Loss of miRNA from ubiquitinated form of HuR . Scheme of the double immunoprecipitation experiment is shown in the left panel . Western blot analysis for HuR in the anti - HA and anti - HuRimmunoprecipitatedFLAG - HuR along with the input of FLAG - HuR used . Lysates of cell expressing the FLAG - HuR and used for FLAGHuR purification is also shown ( right upper panels ) . qRT - PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots ( right lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . Position of ubiquitinated band of FLAG - HuR is marked by arrow .

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

(G) Loss of miRNA from ubiquitinated form of HuR. Scheme of the double immunoprecipitation experiment is shown in the left panel. Western blot analysis for HuR in the anti-HA and anti-HuRimmunoprecipitatedFLAG-HuR along with the input of FLAG-HuR used. Lysates of cell expressing the FLAG-HuR and used for FLAGHuR purification is also shown (right upper panels). qRT-PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots (right lower panel). Data represented as mean+/- s.e.m., n=3. Position of ubiquitinated band of FLAG-HuR is marked by arrow.


( H ) Increase in ubiquitinated form of HuR in Starved Huh7 cells . Levels of ubiquitinated HuR in Fed and Starved ( 5h ) HA - Ub expressing Huh7 cells treated either with ethanol or MG132 ( 20µM ) for same duration . The lysates were immunoprecipitated with HuR and western blotted for HA and HuR . Position of ubiquitinated bands were marked by arrows . The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non - ubiquitinated form were measured and plotted ( mean + / - s . e . m . , n = 3 ) .

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

(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).


( I ) Full length and a truncated version of HA - HuR without the hinge region ( graphical scheme in upper panel ) was used to score the effect of deletion of hinge region on EV associated miR - 122 content measured by qRT - PCR ( bottom panel ) . Western blots to check expression of HuR and its truncated variants are also shown . " * " denotes the HA - HuR band ( left lower panel ) . Data represented as mean + / - s . e . m . , n = 3 .

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

(I) Full length and a truncated version of HA-HuR without the hinge region (graphical scheme in upper panel) was used to score the effect of deletion of hinge region on EV associated miR-122 content measured by qRT-PCR (bottom panel). Western blots to check expression of HuR and its truncated variants are also shown." * " denotes the HA-HuR band (left lower panel). Data represented as mean+/- s.e.m., n=3.


( J ) Ubiquitination status of full length and a truncated version of HA - HuR without the hinge region between RRMII and RRMIII in Huh7 cells . HuR ( full lenght or truncated version ) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA - HuR and ubiquitination . Ubiquinitated HuR bands are marked by arrows . postion of non - ubiquitinated proteins were marked by arrowheads ( upper panel ) . qRT - PCR based estimation of associated miRNA per unit of HA - HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots ( lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR - 122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells .

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

(J) Ubiquitination status of full length and a truncated version of HA-HuR without the hinge region between RRMII and RRMIII in Huh7 cells. HuR (full lenght or truncated version) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA-HuR and ubiquitination. Ubiquinitated HuR bands are marked by arrows. postion of non-ubiquitinated proteins were marked by arrowheads (upper panel). qRT-PCR based estimation of associated miRNA per unit of HA-HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots (lower panel). Data represented as mean+/- s.e.m., n=3. PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR-122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells.


C ) RNA binding - deficient TDP - 43 mutants generated by site - directed substitutions in RRM1 ( F2L ) or in both RRM domains ( F4L ) and substitution of RRM1 and RRM2 with monomeric GFP ( N - GFP - C ) . Phase separation of the mutants without added RNA ( control ) or in the presence A ( GU ) 6 ( 8 μM ) , A ( GU ) 18 ( 2 . 6 μM ) , or A ( CA ) 6 ( 8 μM ) at 150 mM NaCl ( 4 µM protein ) .

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

C) RNA binding-deficient TDP-43 mutants generated by site-directed substitutions in RRM1 (F2L) or in both RRM domains (F4L) and substitution of RRM1 and RRM2 with monomeric GFP (N-GFP-C). Phase separation of the mutants without added RNA (control) or in the presence A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) at 150 mM NaCl (4 µM protein).


B ) TDP - 43 condensates in the presence of the different RNA sequences . TDP - 43 and RNA oligonucleotide concentration is 4 μM and 3 . 9 μM , respectively . Representative images for 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm . C ) LLPS measured by turbidity in the same experimental conditions as panel B . Mean and SD from 3 biological replicates with 2 different protein preparations .

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

B) TDP-43 condensates in the presence of the different RNA sequences. TDP-43 and RNA oligonucleotide concentration is 4 μM and 3.9 μM, respectively. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm. C) LLPS measured by turbidity in the same experimental conditions as panel B. Mean and SD from 3 biological replicates with 2 different protein preparations.


D ) Lysate from HEK 293 cells expressing a GFP - tagged copy of either wild - type ( WT ) or F147 / 149 / 229 / 231L ( F4L ) TDP - 43 was added to a mixture of recombinant WT or F4L TDP - 43 ( 5 . 3 μM , 10 % Cy3 - labeled protein ) and A ( GU ) 6 ( 22 . 8 μM ) , A ( CA ) 18 ( 7 . 6 μM ) , A ( GU ) 18 ( 7 . 6 μM ) , or no RNA control at 250 mM NaCl . Droplets were imaged by brightfield and fluorescence microscopy . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm . E ) LLPS measured by turbidity in the same experimental conditions as panel D . Mean and SD of 4 biological replicates using 2 recombinant protein preparations . Analyzed by one - way ANOVA ( F ( 7 , 24 ) = 69 . 30 , P < 0 . 0001 ) . Sidak ' s multiple comparisons test was used to compare selected groups . * P < 0 . 05 , * * * * P < 0 . 0001 .

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

D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm. E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001.


A ) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or increasing A ( GU ) 18 RNA concentration at 250 mM salt . Representative images from 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm .

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

A) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or increasing A(GU)18 RNA concentration at 250 mM salt. Representative images from 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.


D ) Phase separation of TDP - 43 ( 4 μM ) at increasing salt concentration without RNA or in the presence of increasing A ( GU ) 18 concentration . Scale bars , 10 μm . Inlay added to top right panel for increased visibility , scale bar 1 μm . E ) Phase diagram derived from panel D , denoting either no droplets or droplet formation for each condition .

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

D) Phase separation of TDP-43 (4 μM) at increasing salt concentration without RNA or in the presence of increasing A(GU)18 concentration. Scale bars, 10 μm. Inlay added to top right panel for increased visibility, scale bar 1 μm. E) Phase diagram derived from panel D, denoting either no droplets or droplet formation for each condition.


A ) TDP - 43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or GU - repeat RNA oligonucleotides of increasing length at 250 mM salt . As indicated , RNA concentration varied to maintain a constant total number of TDP - 43 binding sites . Scale bars , 10 μm . B ) The plot shows droplet area quantified as a function of GU - repeat length . Mean and SD of > 300 droplets from 3 biological replicates using 3 different protein preparations . C ) TDP - 43 concentration in the light phase ( Cout ) as a function of GU ( blue ) or CA ( red ) - repeat length quantified by Bradford protein assay . Mean and SD from > 5 biological replicates using 2 different protein preparations . ( 250 mM NaCl , 4 µM TDP - 43 )

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

A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm. B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations. C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)


B ) Phase separation of full - length and TDP - 43 variants ( 4 μM ) mixed with A ( GU ) 30 RNA ( 0 . 8 μM ) or no RNA control at 250 mM NaCl observed by brightfield microscopy . Representative images for 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm .

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

B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.


A ) TDP - 43 ALS - linked amino acid substitutions K181E , A315T / E , A321G , Q331K , M337V , and A382T . Boxed in the C - terminal domain is the α - helical structure within the conserved region ( a . a . 320 - 343 ) . Droplets formed by TDP - 43 wild - type ( WT ) and the different mutants ( 4 μM ) observed by brightfield microscopy in the presence of no RNA control or A ( GU ) 18 RNA ( 3 . 9 μM ) at 250 mM NaCl . Mutations A321G and M337V , showing decreased liquidity of the condensates in the presence of A ( GU ) 18 , are boxed in red . Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V , 2 preparations of A321G , Q331K , K181E and one preparation of A315T / E and A382T . Scale bars , 10 μm .

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

A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.


B ) Phase separation of WT ( 4 μM , 10 % Oregon green - labeled protein ) and M337V ( 4 μM , 10 % Cy - 3 - labeled protein ) observed by brightfield and fluorescence microscopy in the presence of A ( GU ) 18 ( 3 . 9 μM ) at 250 mM NaCl . The middle panel shows mixing of these WT ( 2 μM ) and M337V ( 2 μM ) samples . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-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 O O O 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) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.


A ) Binding of TDP - 43 to GU - rich RNA promotes liquid properties of TDP - 43 condensates , as shown by the fusion of small condensates into larger droplets . This fluidity is impaired by the loss of RNA binding affinity or disease - linked mutations that alter phase separation , such as M337V and A321G , which could explain increased TDP - 43 aggregation .

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

A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.


C , Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials ( TPP : Tetraphenylphosphonium , Et : Ethidium ) , while still retaining partial activity against divalent cations ( Dq : Dequalinium , Ch : Chlorhexidine ) . EV : pBAD empty vector . n = 3 for biological replicates . CFU indicates colony forming units . Image color was inverted from black to white background to enhance clarity .

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

C, Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials (TPP: Tetraphenylphosphonium, Et: Ethidium), while still retaining partial activity against divalent cations (Dq: Dequalinium, Ch:Chlorhexidine). EV: pBAD empty vector. n = 3 for biological replicates. CFU indicates colony forming units. Image color was inverted from black to white background to enhance clarity.


E , Population shift - based titration of QacA - GFP against ICabs yielded an apparent Kd of 65 ± 4 nM for QacA - A4 complex and 100 ± 10 nM for QacA - B7 . n = 3 for independent replicates . Error bars denote S . E . M . and each datapoint represents mean value .

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

E, Population shift-based titration of QacA-GFP against ICabs yielded an apparent Kd of 65 ± 4 nM for QacA-A4 complex and 100 ± 10 nM for QacA-B7. n = 3 for independent replicates. Error bars denote S.E.M. and each datapoint represents mean value.