( H ) Kaplan Meier Curve of human prostate ( n = 499 from 498 TCGA samples ) biochemical recurrence based on primary tumor alterations in a core 8 - IRG signature ( Appendix Fig S7D ) . Patient groups stratified by mRNA z - Score ( RNA - seq V2 RSEM ) ± 1 . 5 . Hazard ratio 1 . 674 ( 95 % confidence interval ( CI ) 2 . 707 to 1 . 035 ) ; * p = 0 . 03 by log - rank test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.


B - F Live confocal images of ddaC clones expressing mCD8 : : GFP driven by ppk - Gal4 at WP stage or 16 h APF . l ( 3 ) 810 and l ( 3 ) 924 mutant ddaC clones ( C , E ) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF . ( D , F ) Introduction of one genomic construct of msps , HN267 / g - msps , rescued dendrite pruning defects in msps810 and msps924 clones . Red arrowheads point to the ddaC somas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.


G Percentages of ddaC clones showing severing defects at 16 h APF .

G Percentages of ddaC clones showing severing defects at 16 h APF.


H Quantitative analysis of unpruned dendrite lengths at 16 h APF .

H Quantitative analysis of unpruned dendrite lengths at 16 h APF.


A Pan - neuronal driver elav - Gal4 was used to co - express GFP - Msps and TACC in postmitotic neurons . elav - Gal4 driven mCD8 : : GFP expression was used as a control . TACC was pulled down by GFP - Msps but not by mCD8 : : GFP . Alpha - tubulin was used as a loading and probing control . Neither mCD8 : : GFP nor GFP - Msps could pull down alpha - tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.


B Endogenous Msps proteins were co - immunoprecipitated with Venus - TACC by GFP beads but not by control IgG beads . Alpha - tubulin was used as loading and probing control .

O O O O O O O O O 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 Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.


C Endogenous Msps or TACC was pulled down by their respective antibodies , and the other protein was simultaneously detected in the immunoprecipitated contents . Alpha - tubulin was used as loading and probing control .

O O O O O O O O O O O O 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 Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.


D - G Confocal images of ddaC neurons expressing control RNAi ( D ) , msps RNAi ( E ) , tacc RNAi ( F ) and αTub84B RNAi ( G ) that were immunostained for TACC at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas .

H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.


I - L Confocal images of ddaC neurons expressing control RNAi ( I ) , msps RNAi ( J ) , tacc RNAi ( K ) and αTub84B RNAi ( L ) that were immunostained for Msps at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas .

M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.


N , O Protein expression of Msps and TACC in brain tissues . ( N ) Larval brains of msps810 / mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading control . The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae.


O Protein expression of Msps and TACC in brain tissues . ( O ) Adult brains of tacc59 / tacc74 transheterozygotes were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading and probing control . The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O Protein expression of Msps and TACC in brain tissues. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.


A - C Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . While expression of control RNAi in ddaC neurons ( A ) did not affect the dendrite morphogenesis nor the pruning processes , knockdown of tacc using RNAi # 1 ( B ) or tacc59 / tacc74 transheterozygous mutant ( C ) severely perturbed the dendrite arborization as well as pruning . Red arrowheads point to the ddaC somas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.


D Percentages of ddaC neurons showing severing defects in ( A - C ) at 16 h APF .

D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.


E Quantitative analysis of unpruned dendrite lengths in ( A - C ) at 16 h APF .

E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.


F - H Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown of tacc and msps via co - expressing tacc RNAi and msps RNAi ( H ) in the ddaC neurons did not enhance the pruning defects , compared to those neurons co - expressing control RNAi and msps RNAi ( F ) . Red arrowheads point to the ddaC somas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.


I Percentages of ddaC neurons showing severing defects in ( F - H ) at 16 h APF .

I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.


J Quantitative analysis of unpruned dendrite lengths in ( F - H ) at 16 h APF .

J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.


Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP , UAS - Nod - lacZ or UAS - Kin - lacZ and immunostained for β - galactosidase at wL3 stages . ( A - C ) Wild - type ddaC neurons ( A ) displayed normal distribution of Nod - lacZ signals in dendrites . In contrast , in msps810 mutants ( B ) and tacc59 / tacc74 mutants ( C ) , ddaC neurons exhibited altered Nod - lacZ distribution patterns , with highly enriched stainings in the somas and decreased signals in the dendrites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites.


Confocal images of ddaC neurons ( D - F ) Kin - lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons ( D ) , whereas part of the Kin - lacZ were mislocalized to dendrites in msps RNAi ( E ) and tacc RNAi ( F ) ddaC neurons . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured . White arrowheads point to dendritically localized Kin - lacZ signals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.


G Percentages of ddaC neurons showing defective Nod - lacZ distribution in mutants or RNAi - expressing neurons .

G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.


H Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma .

O O O O O O O O O O O O O O O O O O O O O O O

H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.


I Percentages of ddaC neurons showing defective Kin - lacZ distribution .

I Percentages of ddaC neurons showing defective Kin-lacZ distribution.


A - F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . EB1 : : GFP expression was driven under Gal44 - 77 . In the control ddaC neurons ( A , D ) , EB1 comets move predominantly in retrograde direction . EB1 comets were largely diminished in msps810 / mspsP mutant neurons ( B ) . Interestingly , in msps810 / mspsP18 mutants ( C ) , tacc RNAi ( E ) and tacc59 / tacc74 mutant ( F ) , increased anterograde comets were detected in ddaC dendrites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.


G - J Quantitative analysis of the percentages of anterograde EB1 : : GFP comets , the track length , the number ( per 30 µm ) and the velocity of the comets in each neuron imaged .

O O O O O O O O O O O O O O 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 Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.


A - F Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP ( Green channel ) , UAS - Nod - lacZ and immunostained for β - galactosidase at wL3 stages . Simultaneous knockdown of klp10A and msps ( B ) significantly restored Nod - lacZ localization in the dendrites , compared to msps and control RNAi ( A ) . ddaC neurons with colchicine treatment ( D ) or with Kat - 60 overexpression ( F ) exhibited perturbed Nod - lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.


G - L Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown klp10A and msps ( H ) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps , control RNAi neurons . Wild type larvae fed with colchicine ( J ) showed pruning defects in comparison with those feed with DMSO ( I ) . ddaC neurons with Kat - 60 overexpression ( L ) exhibited abnormal dendrite arborization and pruning defects . Red arrowheads point to the ddaC somas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.


M , N Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.


O Percentages of ddaC neurons showing severing defects at 16 h APF .

O Percentages of ddaC neurons showing severing defects at 16 h APF.


P Quantitative analysis of unpruned dendrite lengths at 16 h APF .

P Quantitative analysis of unpruned dendrite lengths at 16 h APF.


- F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . ( B ) RNAi knockdown of klp10A in msps810 / mspsP18 mutants restored the retrograde movement pattern of EB1 comets . MT depolymerization or severing induced by Colchicine treatment ( D ) or overexpression of Kat - 60 ( F ) led to mixed MT orientation in ddaC neurons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.


G - J Quantitative analysis of the percentages of anterograde EB1 comets , the track length , number ( per 30 µm ) and the velocity of EB1 comets in each neuron imaged .

O O O O O O O O O O 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 Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.


( A ) p53 - dependent dephosphorylation of Ulk1 at Ser637 . p53 + / + and p53 − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells assessed by propidium iodide ( PI ) staining . α - Tubulin was used as a loading control . A semiquantitative analysis of protein expression is shown in Fig . EV1 .

O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) p53-dependent dephosphorylation of Ulk1 at Ser637. p53+/+ and p53−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells assessed by propidium iodide (PI) staining. α-Tubulin was used as a loading control. A semiquantitative analysis of protein expression is shown in Fig. EV1.


( B , C ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy . PPM1D + / + MEFs and PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells.


( B , C ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy . PPM1D + / + MEFs and PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells . ( C ) Semiquantitative analysis of protein expression is shown ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant . ( D - I ) Suppression of etoposide - induced autophagy in PPM1D − / − MEFs .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean ± SD). *p < 0.05, **p < 0.01, NS: not significant. (D-I) Suppression of etoposide-induced autophagy in PPM1D−/−MEFs.


( D , E ) The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr and then immunostained with an anti - LC3 antibody ( green ) . Representative images are shown in ( D ) . LC3 puncta are observed in etoposide - treated PPM1D + / + MEFs . ( E ) 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 , * * p < 0 . 01 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then immunostained with an anti-LC3 antibody (green). Representative images are shown in (D). LC3 puncta are observed in etoposide-treated PPM1D+/+MEFs. (E) 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, **p < 0.01.


( F , G ) The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr and then analyzed using electron microscopy . Representative images are shown in ( F ) . Many autophagic vacuoles ( arrows ) can be seen in etoposide - treated PPM1D + / + cells ( upper panel ) . " N " indicates the nucleus . Bar = 2 µm . A representative autophagosome ( AP ) and autolysosome ( AL ) are shown in the insets . ( G ) The number of autophagosomes and autolysosomes in each cell were counted ( n > 8 cells ) . Red lines indicate the mean value . * p < 0 . 05 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then analyzed using electron microscopy. Representative images are shown in (F). Many autophagic vacuoles (arrows) can be seen in etoposide-treated PPM1D+/+cells (upper panel). "N" indicates the nucleus. Bar = 2 µm. A representative autophagosome (AP) and autolysosome (AL) are shown in the insets. (G) The number of autophagosomes and autolysosomes in each cell were counted (n > 8 cells). Red lines indicate the mean value. *p < 0.05.


( H , I ) Analysis of autophagic flux . The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr in the presence or absence of bafilomycin A1 ( 10 nM ) , and the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . ( I ) Semiquantitative analyses of protein expression ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, I) Analysis of autophagic flux. The indicated MEFs were treated with etoposide (10 µM) for 6 hr in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses of protein expression (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( A , B ) Effect of stable expression of PPM1D in PPM1D − / − MEFs . PPM1D − / − MEFs and PPM1D - transfected PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . ( B ) Semiquantitative analyses of protein expression in ( A ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of stable expression of PPM1D in PPM1D−/−MEFs. PPM1D−/−MEFs and PPM1D-transfected PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. (B) Semiquantitative analyses of protein expression in (A) (n = 3; mean ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( C , D ) Effect of a PPM1D inhibitor on etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) in the presence or absence of GSK2830371 ( 30 µM ) , and the expression of each protein was examined by western blotting . ( D ) Semiquantitative analysis of protein expression in ( C ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of a PPM1D inhibitor on etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) in the presence or absence of GSK2830371 (30 µM), and the expression of each protein was examined by western blotting. (D) Semiquantitative analysis of protein expression in (C) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( E , F ) Effect of the transient overexpression of PPM1D . PPM1D + / + MEFs were transfected with a PPM1D - Flag plasmid in the presence or absence of bafilomycin A1 ( 10 nM ) , and the expression of each protein was examined by western blotting . ( F ) Semiquantitative analyses of protein expression in ( E ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) Effect of the transient overexpression of PPM1D. PPM1D+/+MEFs were transfected with a PPM1D-Flag plasmid in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. (F) Semiquantitative analyses of protein expression in (E) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( A ) Physical interaction between endogenous Ulk1 and endogenous PPM1D . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) or left untreated for 6 hr . Cells were then lysed and immunoprecipitated with an anti - Ulk1 antibody or a control IgG . Immune complexes and total lysates ( 1 . 8 % input ) were analyzed by western blotting using anti - PPM1D and anti - Ulk1 antibodies .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Physical interaction between endogenous Ulk1 and endogenous PPM1D. PPM1D+/+MEFs were treated with etoposide (10 µM) or left untreated for 6 hr. Cells were then lysed and immunoprecipitated with an anti-Ulk1 antibody or a control IgG. Immune complexes and total lysates (1.8% input) were analyzed by western blotting using anti-PPM1D and anti-Ulk1 antibodies.


( B ) Interaction between endogenous Ulk1 and recombinant PPM1D . Lysates from healthy PPM1D + / + MEFs were incubated with GST - PPM1D or GST . Binding molecules were then analyzed by western blotting using anti - Ulk1 and anti - GST antibodies . " Total lysate " indicates 7 % of the lysates that were incubated with GST fusion protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interaction between endogenous Ulk1 and recombinant PPM1D. Lysates from healthy PPM1D+/+ MEFs were incubated with GST-PPM1D or GST. Binding molecules were then analyzed by western blotting using anti-Ulk1 and anti-GST antibodies. "Total lysate" indicates 7% of the lysates that were incubated with GST fusion protein.


( C ) In vitro Ulk1 dephosphorylation assay . Immunoprecipitated Ulk1 from PPM1D + / + MEF lysates was incubated with GST - PPM1D ( 1 µg ) with or without a phosphatase inhibitor cocktail for 1 hr . Then , the extent of Ulk1 dephosphorylation was analyzed by western blotting using anti - phospho - Ulk1637 and anti - phospho - Ulk1757 antibodies . Semiquantitative analyses are shown in Fig . EV2B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro Ulk1 dephosphorylation assay. Immunoprecipitated Ulk1 from PPM1D+/+ MEF lysates was incubated with GST-PPM1D (1 µg) with or without a phosphatase inhibitor cocktail for 1 hr. Then, the extent of Ulk1 dephosphorylation was analyzed by western blotting using anti-phospho-Ulk1637 and anti-phospho-Ulk1757 antibodies. Semiquantitative analyses are shown in Fig. EV2B.


( D ) The presence of cytosolic PPM1D puncta in etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for 6 hr , immunostained with an anti - PPM1D antibody , and their nuclei stained with Hoechst 33342 ( 50 ng / ml ) . Representative images of anti - PPM1D ( green ; upper panels ) and Hoechst 33342 ( blue ; lower panels ) are shown . Arrowheads indicate cytoplasmic PPM1D puncta .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The presence of cytosolic PPM1D puncta in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with an anti-PPM1D antibody, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (green; upper panels) and Hoechst 33342 (blue; lower panels) are shown. Arrowheads indicate cytoplasmic PPM1D puncta.


( E ) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for 6 hr , immunostained with anti - PPM1D and anti - Ulk1 antibodies , and their nuclei stained with Hoechst 33342 ( 50 ng / ml ) . Representative images of anti - PPM1D ( red ; left ) , anti - Ulk1 ( green ; middle ) , and a merged image ( right ) are shown . Magnified images of the areas within the dashed squares are shown at the bottom . Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with anti-PPM1D and anti-Ulk1 antibodies, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (red; left), anti-Ulk1 (green; middle), and a merged image (right) are shown. Magnified images of the areas within the dashed squares are shown at the bottom. Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1.


( F - J ) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide - induced autophagy . ( F - H ) Ulk1 / Ulk2 DKO MEFs that were stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) . Then , the cell lysates were collected time - dependently , and the expression of each protein was examined by western blotting . Asterisks in the Ulk1 blots are nonspecific bands . Semiquantitative analyses are shown in Fig . EV2E .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (F-H) Ulk1/Ulk2 DKO MEFs that were stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM). Then, the cell lysates were collected time-dependently, and the expression of each protein was examined by western blotting. Asterisks in the Ulk1 blots are nonspecific bands. Semiquantitative analyses are shown in Fig. EV2E.


( F - J ) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide - induced autophagy . ( I , J ) The indicated MEFs were treated with or without etoposide ( 10 µM ) for 6 hr , followed by immunostaining with an anti - LC3 antibody . Representative images are shown in ( I ) . LC3 puncta are markedly observed in DKO MEFs transfected with wild - type HA - Ulk1 . Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A , respectively . ( J ) The population 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 , * * p < 0 . 01 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (I, J) The indicated MEFs were treated with or without etoposide (10 µM) for 6 hr, followed by immunostaining with an anti-LC3 antibody. Representative images are shown in (I). LC3 puncta are markedly observed in DKO MEFs transfected with wild-type HA-Ulk1. Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A, respectively. (J) The population 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, **p < 0.01.


( A - D ) Ulk1 puncta formation was induced by etoposide in a PPM1D - dependent manner . The indicated MEFs were treated with etoposide ( 10 µM ) with or without GSK2830371 ( 20 µM ) for the indicated times , followed by immunostaining with an anti - Ulk1 antibody . Representative images are shown in ( A ) . Ulk1 puncta are observed in etoposide - treated PPM1D + / + MEFs . ( B - D ) The proportion of cells with Ulk1 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Ulk1 puncta formation was induced by etoposide in a PPM1D-dependent manner. The indicated MEFs were treated with etoposide (10 µM) with or without GSK2830371 (20 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images are shown in (A). Ulk1 puncta are observed in etoposide-treated PPM1D+/+MEFs. (B-D) The proportion of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01, NS: not significant.


( 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 O O O O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O 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. (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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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 .

O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O

(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 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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 .

(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 ) .

(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 .

(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 .

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(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 .

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(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 ) .

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(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 ) .

(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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.