F . Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes , showing procollagen accumulation in Fam134bmo fish . β - actin was used as loading control . Bar graph shows quantification of type II procollagen normalized to β - actin . N = 3 biological replicates . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM I-ORGANISM B-TISSUE O O O O O B-GENEPROD O O B-GENEPROD B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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. Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes, showing procollagen accumulation in Fam134bmo fish. β-actin was used as loading control. Bar graph shows quantification of type II procollagen normalized to β-actin. N=3 biological replicates. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.


G . Representative TEM images of medaka fish chondrocytes . Arrows indicated the ER . Insets show ER enlargement in Fam134bmo chondrocyte . N = 3 fish / genotype were analyzed . Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes . N = 29 cells ( Fam134bwt ) and N = 37 cells ( Fam134bmo ) were analyzed . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * * * p < 0 . 0005 . Scale bar 500 nm . N = nucleus , ER = Endoplasmic Reticulum .

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

G. Representative TEM images of medaka fish chondrocytes. Arrows indicated the ER. Insets show ER enlargement in Fam134bmo chondrocyte. N=3 fish/genotype were analyzed. Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes. N=29 cells (Fam134bwt) and N=37 cells (Fam134bmo) were analyzed. Mean +/- standard error of the mean (sem). Student's un-paired T-test ***p<0.0005. Scale bar 500 nm. N=nucleus, ER= Endoplasmic Reticulum.


H . Lateral ( top ) and ventral ( bottom ) projections of medaka fish embryos with indicated genotypes . Scale bar 1mm . Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble . Mean + / - standard error of the mean ( sem ) of at least n = 8 fish / genotype . Student un - paired T - Test * p < 0 . 05 .

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

H. Lateral (top) and ventral (bottom) projections of medaka fish embryos with indicated genotypes. Scale bar 1mm. Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble. Mean +/- standard error of the mean (sem) of at least n=8 fish/genotype. Student un-paired T-Test *p<0.05.


Alcian blue ( cartilage ) ( I ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) cartilage length ( I ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

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

Alcian blue (cartilage) (I) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) cartilage length (I) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


Alizarin Red ( bone ) ( J ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) bone mineralization ( J ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

Alizarin Red (bone) (J) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) bone mineralization (J) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


B . ARPI ( CSS , ENZA , ODM - 201 , 14449 ) decreased , while R1881 increased , SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells . PSA is used as a positive control .

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B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control.


D . SDH repression during acute phase of ENZA stress increased transcription of different AR - regulated genes without altering AR mRNAs levels in LNCaP cells . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using two tailed unpaired Student ' s t - test * , p < 0 . 05 and * * , p < 0 . 01 compared between groups .

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D. SDH repression during acute phase of ENZA stress increased transcription of different AR-regulated genes without altering AR mRNAs levels in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test *, p<0.05 and **, p<0.01 compared between groups.


E - F . Overexpression of SDHA and SDHB subunits decreased AR protein levels ( E ) and AR transactivation ( F ) in LNCaP cells . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s test * , p < 0 . 05 and * * , p < 0 . 01 compared between groups .

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

E-F. Overexpression of SDHA and SDHB subunits decreased AR protein levels (E) and AR transactivation (F) in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05 and **, p<0.01 compared between groups.


C . SDH - repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA following Tukey ' s test for panels C ( end points ) . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between

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

C. SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


E . Treatment with p - p38 inhibitors : SB203580 ( 10 µM ) or Genistein ( 20 µM ) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells ( left panel ) . Bar graph in the right panel shows band intensity normalized to loading control . Data information : ENZA : enzalutamide . Data shown as mean ± SD of three independent experiments . Statistical analysis was performed using one - way ANOVA following Tukey ' s test for panels E . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between

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E. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups


F . . Treatment with 5 µM STO - 609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA / SDHB subunit silencing .

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

F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing.


G . Thapsigargin , DMM , DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4 - AM probe under confocal microscope .

G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.


A - C . Human patient samples demonstrate relative differences in the immunoexpression of AR , SDHA , p - CaMKK2 / p - AMPK , p - p38 , and p - Hsp27 in untreated ( n = 70 ) , neoadjuvant - treated ( NHT ) ( n = 130 ) and CRPC ( n = 24 ) samples . Representative images ( A ) , dot plots ( B ) , compiled bar graph ( C ) support the AR - SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p - CaMKK2 / p - AMPK / p - p38 / p - Hsp27 axis . AR - positive CRPC samples restored SDHA as well as the whole axis . Data information : Data shown as mean ± SEM . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s test . * * , p < 0 . 01 ; * * * , p < 0 . 001 and * * * * , p < 0 . 0001 . Scale bar : 100µm . All the TMAs were constructed from minimum 2 cores per patient .

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

A-C. Human patient samples demonstrate relative differences in the immunoexpression of AR, SDHA, p-CaMKK2/p-AMPK, p-p38, and p-Hsp27 in untreated (n=70), neoadjuvant-treated (NHT) (n=130) and CRPC (n=24) samples. Representative images (A), dot plots (B), compiled bar graph (C) support the AR-SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p-CaMKK2/p-AMPK/p-p38/p-Hsp27 axis. AR-positive CRPC samples restored SDHA as well as the whole axis. Data information: Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **, p<0.01; ***, p<0.001 and ****, p<0.0001. Scale bar: 100µm. All the TMAs were constructed from minimum 2 cores per patient.


D . IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells . Data shown as means ± SEM In vitro data represent three independent experiments . Statistical analysis was performed for the last data point * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 compared between groups , one - way ANOVA followed by Tukey ' s test .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IVM reversed the cell proliferation benefit mediated by SDH repression in LNCaP cells. Data shown as means ± SEM In vitro data represent three independent experiments. Statistical analysis was performed for the last data point *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test.


E . LNCaP xenografts treated with IVM ( n = 10 ) ( 10mg / kg ) demonstrated lower serum PSA values ( upper panel ) and growth of tumours after castration ( lower panel ) compared to vehicle ( n = 10 ) . PSA was measured every week . Data shown as means ±

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

E. LNCaP xenografts treated with IVM (n=10) (10mg/kg) demonstrated lower serum PSA values (upper panel) and growth of tumours after castration (lower panel) compared to vehicle (n=10). PSA was measured every week. Data shown as means ± SEM


F . IVM treatment reduced p - Hsp27 levels and therefore AR protein level , with subsequent p - CAMKK2 axis activation in castrated LNCaP xenografts . Bar graph in the right panel shows quantification of data from western blot normalized to loading control . Data shown as means ±

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

F. IVM treatment reduced p-Hsp27 levels and therefore AR protein level, with subsequent p-CAMKK2 axis activation in castrated LNCaP xenografts. Bar graph in the right panel shows quantification of data from western blot normalized to loading control. Data shown as means ± SD


( a ) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs , sh1 and sh2 , compared to control ( Ctr ) . Left , representative immunoblot . Actin is shown as loading control and full - length blots are shown in Supplementary Figure 21 . Right , amounts of RARα in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown on the left . Values are normalized for actin and expressed as multiples of control ( None ) values ; n = 3 .

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

(a) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs, sh1 and sh2, compared to control (Ctr). Left, representative immunoblot. Actin is shown as loading control and full-length blots are shown in Supplementary Figure 21. Right, amounts of RARα in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown on the left. Values are normalized for actin and expressed as multiples of control (None) values; n = 3.


( b ) Rates of degradation of long - lived proteins in control and RARα ( - ) cells maintained in the presence or absence of serum for 12 h . Values are expressed as percentage of proteolysis ; n = 3 .

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

(b) Rates of degradation of long-lived proteins in control and RARα(-) cells maintained in the presence or absence of serum for 12 h. Values are expressed as percentage of proteolysis; n = 3.


( c , d ) Percentage of degradation due to lysosomes ( c ) and macroautophagy ( d ) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis ( c ) or with 3 - methyladenine ( 3MA ) to block macroautophagy ( d ) . Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors ; n = 3 . In all panels , all values are mean ± s . e . m . , and differences with control are significant for * P 0 . 05 .

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

(c,d) Percentage of degradation due to lysosomes (c) and macroautophagy (d) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis (c) or with 3-methyladenine (3MA) to block macroautophagy (d). Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors; n = 3. In all panels, all values are mean ± s.e.m., and differences with control are significant for *P 0.05.


( a ) Immunoblot for LC3 - II in control mouse fibroblasts ( Ctr ) or those knocked down for RARα ( RARα ( − ) ) maintained in the presence or absence of serum for the indicated times . Where indicated , protease inhibitors ( PI ) against lysosomal proteolysis were added . Actin is shown as a loading control . ' I ' and ' II ' designate different forms of LC3 , as described in the text . ( b ) Amounts of LC3 - II determined by densitometric quantification of immunoblots . Values are expressed as fold change in value relative to serum - supplemented control cells ; n = 4 . ( c ) Ratio of the amounts of LC3 - II in cells treated with protease inhibitors compared to untreated cells ( None ) . Values are expressed as fold change in value relative to untreated cells ; n = 4 .

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

(a) Immunoblot for LC3-II in control mouse fibroblasts (Ctr) or those knocked down for RARα (RARα(−)) maintained in the presence or absence of serum for the indicated times. Where indicated, protease inhibitors (PI) against lysosomal proteolysis were added. Actin is shown as a loading control. 'I' and 'II' designate different forms of LC3, as described in the text. (b) Amounts of LC3-II determined by densitometric quantification of immunoblots. Values are expressed as fold change in value relative to serum-supplemented control cells; n = 4. (c) Ratio of the amounts of LC3-II in cells treated with protease inhibitors compared to untreated cells (None). Values are expressed as fold change in value relative to untreated cells; n = 4.


( d , e ) Autophagic flux in the same cells as in c expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged channel images ; scale bars , 2 μm . Arrows indicate autolysosomes ( red ) . Shown in e are the quantification of the number of autophagosomes ( mCherry and GFP positive ) per cell ( left ) and percentage of autolysosomes ( mCherry positive and GFP negative ; right ) in > 50 cells in at least four different fields . AV , autophagic vacuoles .

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

(d,e) Autophagic flux in the same cells as in c expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged channel images; scale bars, 2 μm. Arrows indicate autolysosomes (red). Shown in e are the quantification of the number of autophagosomes (mCherry and GFP positive) per cell (left) and percentage of autolysosomes (mCherry positive and GFP negative; right) in >50 cells in at least four different fields. AV, autophagic vacuoles.


( f ) Control and RARα ( - ) cells were transfected with the KFERQ - mCherry1 photoactivatable reporter , and after photoactivation they were maintained in medium with or without serum . Left , representative images . Graph shows quantification of the number of puncta per cell in > 50 cells using the CMA reporter in at least four different fields . Nuclei are labeled with DAPI . Scale bars , 10 μm . In b , c , e and f , all values are mean ± s . e . m . Differences with control ( marked with asterisk ) or with serum - supplemented cells ( marked with § ) are significant for P 0 . 05 . Full - field fluorescence images and full - length blots are shown in Supplementary Figures 2 and 21 , respectively .

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

(f) Control and RARα(-) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter, and after photoactivation they were maintained in medium with or without serum. Left, representative images. Graph shows quantification of the number of puncta per cell in >50 cells using the CMA reporter in at least four different fields. Nuclei are labeled with DAPI. Scale bars, 10 μm. In b, c, e and f, all values are mean ± s.e.m. Differences with control (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.05. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 2 and 21, respectively.


( a ) Rates of degradation of long - lived proteins in mouse fibroblasts untreated ( None ) or treated with 40 μM ATRA and maintained in the presence or absence of serum . Values are expressed as a percentage of proteolysis ; n = 3 .

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(a) Rates of degradation of long-lived proteins in mouse fibroblasts untreated (None) or treated with 40 μM ATRA and maintained in the presence or absence of serum. Values are expressed as a percentage of proteolysis; n = 3.


( b ) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h ; n = 3 .

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(b) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h; n = 3.


( c ) Immunoblot for LC3 - II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors ( PI ) . Top , representative immunoblot . Actin is shown as loading control . Bottom , ratio of amount of LC3 - II in cells treated with PI to that in untreated cells . Values are expressed as fold change in value relative to untreated cells ; n = 4 . ' I ' and ' II ' designate different forms of LC3 , as described in the text .

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(c) Immunoblot for LC3-II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors (PI). Top, representative immunoblot. Actin is shown as loading control. Bottom, ratio of amount of LC3-II in cells treated with PI to that in untreated cells. Values are expressed as fold change in value relative to untreated cells; n = 4. 'I' and 'II' designate different forms of LC3, as described in the text.


( d , e ) Autophagic flux in untreated and ATRA - treated cells expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged - channel images . Arrows indicate autolysosomes ( red ) . Scale bar , 2 μm . Shown in e are the number of autophagosomes ( left ) and percentage of autolysosomes ( right ) after quantification of > 50 cells . AV , autophagic vacuoles .

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(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.


( f ) Mouse fibroblasts expressing the KFERQ - mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum . Left , representative images . Nuclei are labeled with DAPI . Scale bars , 10 μm . Graph shows quantification of the number of puncta per cell in > 50 cells . All values in a - c , e and f are mean ± s . e . m . , and differences with untreated ( marked with asterisk ) or with serum - supplemented cells ( marked with § ) are significant for P 0 . 01 . Full - field fluorescence images and full - length blots are shown in Supplementary Figures 3 and 21 , respectively .

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

(f) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum. Left, representative images. Nuclei are labeled with DAPI. Scale bars, 10 μm. Graph shows quantification of the number of puncta per cell in >50 cells. All values in a-c, e and f are mean ± s.e.m., and differences with untreated (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.01. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 3 and 21, respectively.


( a ) Mouse fibroblasts expressing the KFERQ - mCherry1 photoactivatable reporter without ( None ) or with 20 μM of the indicated compounds imaged 16 h after photoactivation . Insets show higher - magnification images . Nuclei are labeled with DAPI . Scale bars , 10 μm . ( b ) Quantification of the effect of increasing concentrations of GR1 on the same cells . Untreated cells and cells treated with 40 μM ATRA are also shown . Representative images are shown in Supplementary Figure 7 . Graph shows the average number of fluorescent puncta per cell , quantified in > 50 cells . All values are mean ± s . e . m .

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(a) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter without (None) or with 20 μM of the indicated compounds imaged 16 h after photoactivation. Insets show higher-magnification images. Nuclei are labeled with DAPI. Scale bars, 10 μm. (b) Quantification of the effect of increasing concentrations of GR1 on the same cells. Untreated cells and cells treated with 40 μM ATRA are also shown. Representative images are shown in Supplementary Figure 7. Graph shows the average number of fluorescent puncta per cell, quantified in >50 cells. All values are mean ± s.e.m.


( c ) Mouse fibroblasts were cotransfected with the human RARα ( hRARα ) receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units ( RLU ) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h .

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(c) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units (RLU) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h .


( d ) Mouse fibroblasts were cotransfected with the human RARα ( hRARα ) receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h . Values are shown as RLU .

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(d) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection.Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU.


( e ) Mouse fibroblasts were cotransfected with the human RXR receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h .

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(e) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h.


( f ) Mouse fibroblasts were cotransfected with the human RXR receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h . Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h . Values are shown as RLU . Values in c - f are mean ± s . e . m . ; n = 4 - 6 .

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(f) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h. Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU. Values in c-f are mean ± s.e.m.; n = 4-6.


( g ) Left , immunoblot for LC3 - II in cells treated with 20 μM of the retinoid derivatives and protease inhibitors ( PIs ) , as labeled . Actin is shown as loading control , and full - length blots are shown in Supplementary Figure 21 . The amount of LC3 - II in untreated cells , expressed as fold change relative to control ( middle ) , and the increase in LC3 - II after PI treatment ( LC3 - II flux , right ) , expressed as fold change , were calculated from the densitometric quantification of immunoblots . Values are mean ± s . e . m . ; n = 3 .

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(g) Left, immunoblot for LC3-II in cells treated with 20 μM of the retinoid derivatives and protease inhibitors (PIs), as labeled. Actin is shown as loading control, and full-length blots are shown in Supplementary Figure 21. The amount of LC3-II in untreated cells, expressed as fold change relative to control (middle), and the increase in LC3-II after PI treatment (LC3-II flux, right), expressed as fold change, were calculated from the densitometric quantification of immunoblots. Values are mean ± s.e.m.; n = 3.


( a ) Rates of degradation of long - lived proteins in control mouse fibroblasts or RARα ( − ) or LAMP - 2A ( − ) ( L - 2A ( − ) ) cells left untreated ( None ) or treated with 20 μM of the indicated compounds . Values are expressed as the fold change in proteolytic rate relative to the rate in untreated cells for each group ; n = 3 .

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(a) Rates of degradation of long-lived proteins in control mouse fibroblasts or RARα(−) or LAMP-2A(−) (L-2A(−)) cells left untreated (None) or treated with 20 μM of the indicated compounds. Values are expressed as the fold change in proteolytic rate relative to the rate in untreated cells for each group; n = 3.


( b - d ) Control mouse fibroblasts or RARα ( − ) , LAMP - 2A ( − ) or LAMP - 2B ( − ) ( L - 2B ( − ) ) cells were transfected with the KFERQ - mCherry1 photoactivatable reporter with or without the indicated compounds ( 20 μM ) . In b are shown the representative fields and high - magnification images ( insets ) for GR1 . Nuclei are labeled with DAPI . Representative fields for GR2 and AR7 are shown in Supplementary Figure 13 . Scale bars , 10 μm . ( c , d ) Average number of fluorescent puncta per cell quantified in > 50 cells in at least four different fields in control cells and RAR ( − ) ( c ) or LAMP - 2B ( − ) ( d ) cells . No puncta were detected in LAMP - 2A ( − ) cells .

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(b-d) Control mouse fibroblasts or RARα(−), LAMP-2A(−) or LAMP-2B(−) (L-2B(−)) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter with or without the indicated compounds (20 μM). In b are shown the representative fields and high-magnification images (insets) for GR1. Nuclei are labeled with DAPI. Representative fields for GR2 and AR7 are shown in Supplementary Figure 13. Scale bars, 10 μm. (c,d) Average number of fluorescent puncta per cell quantified in >50 cells in at least four different fields in control cells and RAR(−) (c) or LAMP-2B(−) (d) cells. No puncta were detected in LAMP-2A(−) cells.


( e ) Mouse fibroblasts transfected with the KFERQ - mCherry1 photoactivatable reporter with the indicated concentrations of AR7 or GR1 or with both compounds to reach the same final concentration , as indicated . Top , representative fields and high - magnification images ( insets ) . Nuclei are labeled with DAPI . Bottom , quantification of the number of fluorescent puncta per field in each condition . Scale bars , 10 μm . In a , c , d and e , values are mean ± s . e . m . ; n > 50 cells . Differences with untreated samples ( marked with asterisk ) or between single and combined treatments ( § ) are significant for P 0 . 01 .

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(e) Mouse fibroblasts transfected with the KFERQ-mCherry1 photoactivatable reporter with the indicated concentrations of AR7 or GR1 or with both compounds to reach the same final concentration, as indicated. Top, representative fields and high-magnification images (insets). Nuclei are labeled with DAPI. Bottom, quantification of the number of fluorescent puncta per field in each condition. Scale bars, 10 μm. In a, c, d and e, values are mean ± s.e.m.; n > 50 cells. Differences with untreated samples (marked with asterisk) or between single and combined treatments (§) are significant for P 0.01.


( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and lysosomes ( Lys ) isolated from cells untreated ( None ) or treated for 12 h with 20 μM of the indicated compounds .

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(a) Immunoblot for the indicated proteins in homogenates (Hom) and lysosomes (Lys) isolated from cells untreated (None) or treated for 12 h with 20 μM of the indicated compounds.


( b ) mRNA levels of LAMP - 2A in control mouse fibroblasts ( left ) or RARα ( − ) cells ( right ) treated with AR7 or paraquat ( PQ ) as in a ; n = 4 - 5 . Values are presented as fold change in mRNA relative to untreated cells .

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(b) mRNA levels of LAMP-2A in control mouse fibroblasts (left) or RARα(−) cells (right) treated with AR7 or paraquat (PQ) as in a; n = 4-5. Values are presented as fold change in mRNA relative to untreated cells.


( c ) Cellular viability of control ( left ) or LAMP - 2A ( − ) ( right ) fibroblasts exposed to 2 mM or 0 . 5 mM paraquat , respectively , and treated with the indicated compounds for 12 h before or after the paraquat treatment ; n = 3 .

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(c) Cellular viability of control (left) or LAMP-2A(−) (right) fibroblasts exposed to 2 mM or 0.5 mM paraquat, respectively, and treated with the indicated compounds for 12 h before or after the paraquat treatment; n = 3.


( d ) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α - synuclein and left untreated ( None ) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7 ; n = 3 . In b - d , all values are mean ± s . e . m . Differences with cells that are untreated ( marked with asterisk ) or those treated only with paraquat ( § ) were significant for P 0 . 001 . Full - length blots are shown in Supplementary Figure 21 .

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

(d) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α-synuclein and left untreated (None) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7; n = 3. In b-d, all values are mean ± s.e.m. Differences with cells that are untreated (marked with asterisk) or those treated only with paraquat (§) were significant for P 0.001. Full-length blots are shown in Supplementary Figure 21.


( e ) Immunoblot for α - synuclein ( α - syn ) in the same cells as in d . Top , higher - exposure blot to highlight oligomeric ( oligo ) species . Asterisk denotes nonspecific band . M , monomer ; MW , molecular weight .

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(e) Immunoblot for α-synuclein (α-syn) in the same cells as in d. Top, higher-exposure blot to highlight oligomeric (oligo) species. Asterisk denotes nonspecific band. M, monomer; MW, molecular weight.


A . The Armi complex immunoisolated from OSCs using anti - Armi antibodies contains Yb , SoYb , and Vret .

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A. The Armi complex immunoisolated from OSCs using anti-Armi antibodies contains Yb, SoYb, and Vret.


B . The Vret complex immunoisolated from OSCs using anti - Vret antibodies contains Yb , Armi , and SoYb .

B. The Vret complex immunoisolated from OSCs using anti-Vret antibodies contains Yb, Armi, and SoYb.


C . Silver staining of protein components in the Vret complex The lane α - Vret ( left ) contained only the anti - Vret antibodies used in these experiments .

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C. Silver staining of protein components in the Vret complex The lane α-Vret (left) contained only the anti-Vret antibodies used in these experiments.


D . Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment ( kd ) . Even without the association of Yb , Armi , SoYb , and Vret associated . Yb and Armi remained bound to each other upon SoYb or Vret depletion . The level of SoYb ( input ) decreased upon Vret depletion . Likewise , the level of Vret ( input ) decreased upon SoYb depletion .

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D. Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment (kd). Even without the association of Yb, Armi, SoYb, and Vret associated. Yb and Armi remained bound to each other upon SoYb or Vret depletion. The level of SoYb (input) decreased upon Vret depletion. Likewise, the level of Vret (input) decreased upon SoYb depletion.


E . Upon Armi depletion , SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb .

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E. Upon Armi depletion, SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb.


F . Immunofluorescence analyses in OSCs . Yb bodies disappeared upon Yb depletion , which led Armi , SoYb and Vret to be localized in the cytosol . Yb bodies were detected in OSCs upon Armi depletion , but SoYb and Vret localized in the cytosol . Armi was detected at Yb bodies upon SoYb or Vret depletion . Yb bodies are indicated by white arrows . Nuclei are shown in blue . Scale bar : 2 μm .

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F. Immunofluorescence analyses in OSCs. Yb bodies disappeared upon Yb depletion, which led Armi, SoYb and Vret to be localized in the cytosol. Yb bodies were detected in OSCs upon Armi depletion, but SoYb and Vret localized in the cytosol. Armi was detected at Yb bodies upon SoYb or Vret depletion. Yb bodies are indicated by white arrows. Nuclei are shown in blue. Scale bar: 2 μm.


B . In Yb - lacking OSCs ( Yb kd ) , WT Yb and ∆ eTud bound to Armi but ∆ eTud did not . Note that WT Yb and both mutants were engineered to be RNAi - resistant by mutations . β - gal was used as a negative control .

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B. In Yb-lacking OSCs (Yb kd), WT Yb and ∆eTud bound to Armi but ∆eTud did not. Note that WT Yb and both mutants were engineered to be RNAi-resistant by mutations. β-gal was used as a negative control.


C . In normal OSCs , WT Yb and ∆ eTud bound with Yb but ∆ Hel - C did not .

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C. In normal OSCs, WT Yb and ∆eTud bound with Yb but ∆Hel-C did not.


D . Flag - Hel - C copurified with Myc - Hel - C but not with BSD ( negative control ) .

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D. Flag-Hel-C copurified with Myc-Hel-C but not with BSD (negative control).


E . Subcellular localization of WT Yb and deletion mutants ∆ Hel - C and ∆ eTud . Myc - Yb is shown in green . Endogenous Armi is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 5 μm . Western blotting ( right ) showing WT Yb and deletion mutants ∆ Hel - C and ∆ eTud expressed in Yb - lacking OSCs .

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E. Subcellular localization of WT Yb and deletion mutants ∆Hel-C and ∆eTud. Myc-Yb is shown in green. Endogenous Armi is shown in red. DAPI shows nuclei (blue). Scale bar: 5 μm. Western blotting (right) showing WT Yb and deletion mutants ∆Hel-C and ∆eTud expressed in Yb-lacking OSCs.


F . CLIP shows that WT Yb and ∆ Hel - C bind RNA in vivo but ∆ eTud does not .

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F. CLIP shows that WT Yb and ∆Hel-C bind RNA in vivo but ∆eTud does not.


A . Rescue experiments showing that ∆ Hel - C but not ∆ eTud restored piRNA biogenesis attenuated by endogenous Yb depletion . Flag - Piwi was expressed upon endogenous Yb depletion .

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A. Rescue experiments showing that ∆Hel-C but not ∆eTud restored piRNA biogenesis attenuated by endogenous Yb depletion. Flag-Piwi was expressed upon endogenous Yb depletion.


B . The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments . p values were calculated by t - test . * : p < 0 . 05 .

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B. The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments. p values were calculated by t-test. *: p < 0.05.


C . Mapping of flam - piRNAs produced in the presence and absence of the Hel - C domain in OSCs . D . Mapping of tj - piRNAs produced in the presence and absence of the Hel - C domain in OSCs .

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C. Mapping of flam-piRNAs produced in the presence and absence of the Hel-C domain in OSCs. D. Mapping of tj-piRNAs produced in the presence and absence of the Hel-C domain in OSCs.


E . Scatterplot showing changes in the abundances of transposon - repressing piRNAs and non - transposon - repressing piRNAs in the presence and absence of the Hel - C domain . Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation , respectively .

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E. Scatterplot showing changes in the abundances of transposon-repressing piRNAs and non-transposon-repressing piRNAs in the presence and absence of the Hel-C domain. Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation, respectively.


F . The relative enrichments of RNAs coimmunopurified with WT Yb and ∆ Hel - C . Bars and error bars represent means ± SEM values of three independent experiments . In addition to tj , three genic piRNA sources that were characterized in previous studies ( dm / Myc [ 59 ] , c - Fos / kay [ 60 ] , and CG9257 [ 34 ] ) are also shown . rp49 was used as a negative control . p value was calculated by t - test . * * * : p < 0 . 001 .

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F. The relative enrichments of RNAs coimmunopurified with WT Yb and ∆Hel-C. Bars and error bars represent means ± SEM values of three independent experiments. In addition to tj, three genic piRNA sources that were characterized in previous studies (dm/Myc [59], c-Fos/kay [60], and CG9257 [34]) are also shown. rp49 was used as a negative control. p value was calculated by t-test. ***: p < 0.001.


A . Yb bodies ( Yb in green ) disappeared after 1 , 6 - hexanediol treatment of OSCs , as did P bodies ( Dcp1 in red ) . Nuclei are shown in blue . Scale bar : 5 μm .

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A. Yb bodies (Yb in green) disappeared after 1,6-hexanediol treatment of OSCs, as did P bodies (Dcp1 in red). Nuclei are shown in blue. Scale bar: 5 μm.


B . Snapshots of live imaging of GFP - Yb expressed in OSCs Fusion - and fission - like dynamics of Yb bodies were observed . Saturated pixels are shown in red . Scale bar : 0 . 5 μm .

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B. Snapshots of live imaging of GFP-Yb expressed in OSCs Fusion- and fission-like dynamics of Yb bodies were observed. Saturated pixels are shown in red. Scale bar: 0.5 μm.


C . Yb Y23A and F129A mutants ( green ) impaired Yb body formation . Nuclei are shown in blue . Scale bar : 5 μm .

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C. Yb Y23A and F129A mutants (green) impaired Yb body formation. Nuclei are shown in blue. Scale bar: 5 μm.


D . Yb Y23A and F129A mutants failed to produce flam - piRNAs . miR - 310 was used as a loading control .

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D. Yb Y23A and F129A mutants failed to produce flam-piRNAs. miR-310 was used as a loading control.


E . Mapping of flam - and tj - piRNAs produced in the flamKG mutant and heterozygote ovaries .

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E. Mapping of flam- and tj-piRNAs produced in the flamKG mutant and heterozygote ovaries.


A Phenotypes of WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings on MS medium with 2 % sucrose . Scale bars = 0 . 5 cm .

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A Phenotypes of WT, dja6, dja5, dja6 dja5 and RNAi seedlings on MS medium with 2% sucrose. Scale bars = 0.5 cm.


B , C Phenotypes and chlorophyll fluorescence images of 6 - week - old WT , dja6 dja5 and complemented plants ( B ) on MS medium with 2 % sucrose , scale bars = 0 . 5 cm ; WT , dja6 , dja5 and RNAi mutants ( C ) grown on soil , scale bars = 1 cm . Fluorescences in ( B ) and ( C ) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right .

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B,C Phenotypes and chlorophyll fluorescence images of 6-week-old WT, dja6 dja5 and complemented plants (B) on MS medium with 2% sucrose, scale bars = 0.5 cm; WT, dja6, dja5 and RNAi mutants (C) grown on soil, scale bars = 1 cm. Fluorescences in (B) and (C) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right.


D Chlorophyll content of total leaves in 6 - week - old WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings . FW , fresh weight . Data are the means ± SEM ( n = 7 biological replicates ) .

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D Chlorophyll content of total leaves in 6-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings. FW, fresh weight. Data are the means ± SEM (n = 7 biological replicates).


E The ultrastructure of plastids from WT , dja6 , dja5 , dja6 dja5 and RNAi mutants . T , thylakoids . Scale bars = 0 . 2 μm .

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E The ultrastructure of plastids from WT, dja6, dja5, dja6 dja5 and RNAi mutants. T, thylakoids. Scale bars = 0.2 μm.


A , B Total protein was extracted from 4 - week - old WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings , respectively . WT and dja6 dja5 used in ( A ) were grown on MS medium with 2 % sucrose , while WT , dja6 , dja5 and RNAi mutants used in ( B ) were grown on soil . 10 μg protein was loaded , except for detection with anti - SUFE1 and anti - SUFA antiserum , for which 25 μg were loaded . Specific bands were identified by immunoblotting and by their molecular weight , and are indicated by asterisks . Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right , respectively . The Fe - S types of Fe - S proteins are shown in parentheses . Actin served as controls to normalize protein levels . For each protein , three independent biological replicates were performed and a representative one is shown .

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A, B Total protein was extracted from 4-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings, respectively. WT and dja6 dja5 used in (A) were grown on MS medium with 2% sucrose, while WT, dja6, dja5 and RNAi mutants used in (B) were grown on soil. 10 μg protein was loaded, except for detection with anti-SUFE1 and anti-SUFA antiserum, for which 25 μg were loaded. Specific bands were identified by immunoblotting and by their molecular weight, and are indicated by asterisks. Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right, respectively. The Fe-S types of Fe-S proteins are shown in parentheses. Actin served as controls to normalize protein levels. For each protein, three independent biological replicates were performed and a representative one is shown.


A - C Metal concentrations in the WT , dja6 dja5 and RNAi mutants . Fe concentrations in total leaves ( A and B ) and total chloroplast ( C ) from 4 - week - old WT , dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry , and are shown as means ± SEM ( n = 3 biological replicates ) of μg g - 1 dry weight ( DW ) . WT and RNAi mutants used in ( A and C ) were grown on soil , while WT and dja6 dja5 seedlings used in ( B ) were grown on MS medium with 2 % sucrose . Similar results were obtained in two additional independent biological experiments . Asterisks indicate significant differences from the value of WT ( two - sample Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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A-C Metal concentrations in the WT, dja6 dja5 and RNAi mutants. Fe concentrations in total leaves (A and B) and total chloroplast (C) from 4-week-old WT, dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry, and are shown as means ± SEM (n = 3 biological replicates) of μg g-1 dry weight (DW). WT and RNAi mutants used in (A and C) were grown on soil, while WT and dja6 dja5 seedlings used in (B) were grown on MS medium with 2% sucrose. Similar results were obtained in two additional independent biological experiments. Asterisks indicate significant differences from the value of WT (two-sample Student's t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).


D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant . Statistical analysis was performed using Student ' s t - test . Data on the X - axis represent the Log2 - ratio of FC ( fold change ) between the absolute abundances of transcripts identified from dja6 dja5 against WT . The Y - axis represents the Log10 of FDR ( false discovery rate ) . All DEGs ( differentially expressed genes ) with FDR < 0 . 01 and FC > 2 are marked in grey , while all iron - related DEGs with FDR < 0 . 01 and FC > 2 are marked in red . The iron - related DEGs labeled with red dots within the black boxes are referred to in the text . All data are based on three biological replicates .

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

D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant. Statistical analysis was performed using Student's t-test. Data on the X-axis represent the Log2-ratio of FC (fold change) between the absolute abundances of transcripts identified from dja6 dja5 against WT. The Y-axis represents the Log10 of FDR (false discovery rate). All DEGs (differentially expressed genes) with FDR < 0.01 and FC > 2 are marked in grey, while all iron-related DEGs with FDR < 0.01 and FC > 2 are marked in red. The iron-related DEGs labeled with red dots within the black boxes are referred to in the text. All data are based on three biological replicates.


D DJA6 can bind Fe2 + in chloroplast . Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs , respectively . The untransformed protoplasts and protoplasts transformed with non - fused EBFP proteins were used as controls . The protoplasts were further stained by FeRhoNoxTM - 1 ( red ) to visualize the cellular distribution of Fe2 + . Green , EBFP fluorescence signals ; magenta , chloroplast autofluorescence signals ; yellow , regions where the EBFP and FeRhoNoxTM - 1 fluorescence signals merged . The speckles which DJA6 - EBFP co - localizes with FeRhoNoxTM - 1 are indicated by white arrows . Scale bars = 5 μm .

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D DJA6 can bind Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and FeRhoNoxTM-1 fluorescence signals merged. The speckles which DJA6-EBFP co-localizes with FeRhoNoxTM-1 are indicated by white arrows. Scale bars = 5 μm.


B Co - IP assays for the interactions of DJA6 with SUFE1 and SUFC . Total protein extracts of protoplasts co - transfected with designated constructs were subjected to immunoprecipitation , and then analyzed by immunoblotting with antibodies indicated on the right . Input ( left lane ) indicates that 100 μg total protein was loaded on the gel .

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B Co-IP assays for the interactions of DJA6 with SUFE1 and SUFC. Total protein extracts of protoplasts co-transfected with designated constructs were subjected to immunoprecipitation, and then analyzed by immunoblotting with antibodies indicated on the right. Input (left lane) indicates that 100 μg total protein was loaded on the gel.


G , H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts . Scale bars = 10 μm .

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G, H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Scale bars = 10 μm.


C Phenotypes and chlorophyll fluorescence images of 6 - week - old WT and 35Spro : SynDJA6 - FLAG / dja6 dja5 transgenic plants grown on soil . Scale bars = 1 cm .

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C Phenotypes and chlorophyll fluorescence images of 6-week-old WT and 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants grown on soil. Scale bars = 1 cm.


D Expression of FLAG - tagged SynDJA6 in 35Spro : SynDJA6 - FLAG / dja6 dja5 transgenic plants . Total leaf extracts ( 100 μg proteins ) from WT and transgenic seedlings were loaded . FLAG - tagged SynDJA6 is indicated by asterisk .

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D Expression of FLAG-tagged SynDJA6 in 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants. Total leaf extracts (100 μg proteins) from WT and transgenic seedlings were loaded. FLAG-tagged SynDJA6 is indicated by asterisk.


Tissue microarrays ( TMAs ) from primary PCa ( n = 132 ) and CRPC ( n = 148 ) were stained via immunohistochemistry for poly ( ADP - ribose ) ( PAR ) , and scored by a clinical pathologist ( T . Parsons ) for intensity ( 0 - 3 ) and percentage ( 0 - 3 ) PAR score was generated via the equation : ( intensity x 1 ) + ( percentage x 2 ) . PAR scores were compared between primary and CRPC . * * * * = p value < 0 . 0001 by Chi - square

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Tissue microarrays (TMAs) from primary PCa (n=132) and CRPC (n=148) were stained via immunohistochemistry for poly(ADP-ribose) (PAR), and scored by a clinical pathologist (T. Parsons) for intensity (0-3) and percentage (0-3) PAR score was generated via the equation: (intensity x 1) + (percentage x 2). PAR scores were compared between primary and CRPC. ****=p value<0.0001 by Chi-square test


Manual PAR scores were divided in to quartiles and then were compared to progression - free survival in the CRPC TMAs . * = p < 0 . 05 , ns = not statistically significant by Log - rank ( Mantel - Cox )

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Manual PAR scores were divided in to quartiles and then were compared to progression-free survival in the CRPC TMAs. *=p<0.05, ns=not statistically significant by Log-rank (Mantel-Cox)


Representative image of one TMA core after multiplex fluorescent IHC for γH2AX ( green ) , PAR ( red ) , PARP - 1 ( purple ) , with DNA ( blue ) . Top Right : Insets of parent image at left . Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation ( boxed areas ) . Bottom Left : Percent positive staining for PAR for the entirety of each TMA cohort . Bottom Middle : Percent positive staining for PARP - 1 . Bottom right : γH2AX for the entirety of each TMA cohort . Data was considered after a median intensity cutoff , and analyzed for statistical significance using two - tailed Student " s t - test for PAR , PARP - 1 , and γH2AX , respectively . Exact p values are

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Representative image of one TMA core after multiplex fluorescent IHC for γH2AX (green), PAR (red), PARP-1 (purple), with DNA (blue). Top Right: Insets of parent image at left. Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation (boxed areas). Bottom Left: Percent positive staining for PAR for the entirety of each TMA cohort. Bottom Middle: Percent positive staining for PARP-1. Bottom right: γH2AX for the entirety of each TMA cohort. Data was considered after a median intensity cutoff, and analyzed for statistical significance using two-tailed Student"s t-test for PAR, PARP-1, and γH2AX, respectively. Exact p values are indi


Two - tailed Spearman correlation test between PAR and γH2AX ( % positive with a median intensity cut - off ) . Exact p values are indicated when

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Two-tailed Spearman correlation test between PAR and γH2AX (% positive with a median intensity cut-off). Exact p values are indicated when available


Top , left : Schematic representing the conditions utilized for transcriptomic analyses ( n = 2 ) of HT - sensitive LNCaP cells . Cells were deprived of hormones for 72 hours , followed by either treatment with 2 . 5uM veliparib ( PARPi ) or vehicle control ( DMSO ) for 1 hour , then subsequently treated with either 1nM DHT or vehicle control ( EtOH ) for 16 hours . Top , right : Immunoblot with the indicated antisera . Bottom : Volcano plots of transcripts found to be differentially regulated by DHT v . EtOH ( left ) or DHT v . PARPi followed by DHT ( right ) . Red dots indicate transcripts that were both statistically significantly altered ( p < 0 . 05 ) and more than 1 . 5 - fold

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Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of HT-sensitive LNCaP cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for 1 hour, then subsequently treated with either 1nM DHT or vehicle control (EtOH) for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated by DHT v. EtOH (left) or DHT v. PARPi followed by DHT (right). Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed


Top , left : Schematic representing the conditions utilized for transcriptomic analyses ( n = 2 ) of CRPC C4 - 2 cells . Cells were deprived of hormones for 72 hours , followed by either treatment with 2 . 5uM veliparib ( PARPi ) or vehicle control ( DMSO ) for for 16 hours . Top , right : Immunoblot with the indicated antisera . Bottom : Volcano plots of transcripts found to be differentially regulated PARPi v . vehicle control . Red dots indicate transcripts that were both statistically significantly altered ( p < 0 . 05 ) and more than 1 . 5 - fold

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Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of CRPC C4-2 cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated PARPi v. vehicle control. Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed


Genes found to be down - regulated by PARPi as described above ( p value < 0 . 05 , 1 . 5 - fold change ) in either HT - sensitive cells ( left ) or CRPC cells ( right ) were queried against the expression of these genes in the Grasso et al data set in Oncomine . Benign = grey , primary PCa = blue , metastases = orange . Boxplot was generated using the mean expression of the PARPi down - regulated genes in the indicated data sets . Statistical significance determined by two - tailed Student " s

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

Genes found to be down-regulated by PARPi as described above (p value < 0.05, 1.5-fold change) in either HT-sensitive cells (left) or CRPC cells (right) were queried against the expression of these genes in the Grasso et al data set in Oncomine. Benign = grey, primary PCa = blue, metastases = orange. Boxplot was generated using the mean expression of the PARPi down-regulated genes in the indicated data sets. Statistical significance determined by two-tailed Student"s t


Indicated cell lines were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological experiments . Statistical significance was determine by two - tailed Student " s t test where * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . LNCaP : E2F1 , p = 0 . 0159 ; PCNA , p = 0 . 0217 ; MCM7 , p = 4 . 0936e - 6 ; CCNA2 , p = 0 . 0005 . C4 - 2 : E2F1 , p = 0 . 0074 ; PCNA , p = 0 . 1258 ; MCM7 , p = 3 . 7471e - 5 ; CCNA2 , p =

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

Indicated cell lines were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determine by two-tailed Student"s t test where *=p<0.05, **=p<0.01,***=p<0.001, ****=p<0.0001. LNCaP: E2F1, p=0.0159; PCNA, p=0.0217; MCM7, p=4.0936e-6; CCNA2, p=0.0005. C4-2: E2F1, p=0.0074; PCNA, p=0.1258; MCM7, p=3.7471e-5; CCNA2, p=0


ChIP - qPCR after C4 - 2 cells were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological

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ChIP-qPCR after C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments


Athymic nude mice were injected with C4 - 2 cell mixed with matrigel . Once tumors became 100mm3 , mice were treated with either vehicle control or veliparib . 72 hours later , tumors were harvested , RNA was isolated and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples , + / - standard deviation of three independent xenograft

O O B-ORGANISM O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-DISEASE O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-DISEASE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY

Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors


Prostatectomy tissue ( n = 6 ) was cultured as previously described , and treated with either vehicle control or veliparib for six days . RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar

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Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar color


Indicated cell lines were treated as depicted in Figure 2 , and labeled with bromodeoxyuridine ( BrdU ) , harvested at indicated time points and utilized for FACS

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Indicated cell lines were treated as depicted in Figure 2, and labeled with bromodeoxyuridine (BrdU), harvested at indicated time points and utilized for FACS analyses


Left : Data generated as described above in Figure 2 was used to generate a heatmap of homologous recombination ( HR ) gene expression after the indicated treatment regimens . Middle : Selected GSEA MSigDB Oncogenic Signature pathways are shown . Right : Data generated as described above in Figure 2 was compared to a previously described HR deficiency transcriptional profile ( Peng et al . , Nature Communications , 2014 ) . This profile was derived by independently silencing either BRCA1 , RAD51 , or BRIP1 , followed by transcriptional analyses . The union of these three data sets was used to generate the signature . Cut - offs for comparison were a p value < 0 . 05 , and fold change of 1 . 5 . Venn diagrams show the overlapping and non - overlapping genes of both down - ( top ) and up - regulated ( bottom ) genes in the previously - defined HR deficiency signature , and the PARPi - responsive

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

Left: Data generated as described above in Figure 2 was used to generate a heatmap of homologous recombination (HR) gene expression after the indicated treatment regimens. Middle: Selected GSEA MSigDB Oncogenic Signature pathways are shown. Right: Data generated as described above in Figure 2 was compared to a previously described HR deficiency transcriptional profile (Peng et al., Nature Communications, 2014). This profile was derived by independently silencing either BRCA1, RAD51, or BRIP1, followed by transcriptional analyses. The union of these three data sets was used to generate the signature. Cut-offs for comparison were a p value<0.05, and fold change of 1.5. Venn diagrams show the overlapping and non-overlapping genes of both down- (top) and up-regulated (bottom) genes in the previously-defined HR deficiency signature, and the PARPi-responsive transcriptome


Left : C4 - 2 cells were treated as depicted in Figure 2 . Data are depicted as mean + / - standard deviation of three independent biological experiments . Statistical significance was determined by two - tailed Student " s t test where * = p < 0 . 05 , * * = p < 0 . 01 . BRCA2 , p = 0 . 0046 ; RAD51 , p = 0 . 0151 ; XRCC3 , p = 0 . 0341 ; TOP3A , p = 0 . 04988 . Right : C4 - 2 cells were treated as depicted in Figure 2 and immunoblotted with the indicated antisera . Quantifications shown below

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

Left: C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determined by two-tailed Student"s t test where *=p<0.05, **=p<0.01. BRCA2, p=0.0046; RAD51, p=0.0151; XRCC3, p=0.0341; TOP3A, p=0.04988. Right: C4-2 cells were treated as depicted in Figure 2 and immunoblotted with the indicated antisera. Quantifications shown below each


Prostatectomy tissue ( n = 6 ) was cultured as previously described , and treated with either vehicle control or veliparib for six days . RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples . Each individual tissue is depicted by a separate bar color . Statistical analyses were performed by Wilcoxon signed rank

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

Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples. Each individual tissue is depicted by a separate bar color. Statistical analyses were performed by Wilcoxon signed rank test


C4 - 2 cells were treated as depicted in Figure 2 . ChIP was performed using the indicated antisera , and the subsequent DNA was isolated and used in qPCR reaction using primers designed to amplify the indicated genomic loci : BRCA2 enhancer , RAD51 promoter , or TOP3A

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C4-2 cells were treated as depicted in Figure 2. ChIP was performed using the indicated antisera, and the subsequent DNA was isolated and used in qPCR reaction using primers designed to amplify the indicated genomic loci: BRCA2 enhancer, RAD51 promoter, or TOP3A promoter


C4 - 2 cells treated with 2 . 5uM veliparib ( Vel . ) or vehicle control ( Veh . ) for 24 hours . Cells were then harvested , lysed , and differentially centrifuged as described in the material and methods section , resulting in a soluble fraction ( Sol . ) ( GAPDH serves as control ) or a chromatin - tethered fraction ( Teth . ) ( histone H4 serves as control ) . Immunoblots were performed for the indicated

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C4-2 cells treated with 2.5uM veliparib (Vel.) or vehicle control (Veh.) for 24 hours. Cells were then harvested, lysed, and differentially centrifuged as described in the material and methods section, resulting in a soluble fraction (Sol.) (GAPDH serves as control) or a chromatin-tethered fraction (Teth.) (histone H4 serves as control). Immunoblots were performed for the indicated proteins


Expression levels of indicated HR pathway genes in primary PCa vs normal patient samples . Violin plots represent FPKM normalized counts obtained from matched tumor and normal RNA - Seq data from TCGA ( n = 52 ) with p - values generated using paired t -

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Expression levels of indicated HR pathway genes in primary PCa vs normal patient samples. Violin plots represent FPKM normalized counts obtained from matched tumor and normal RNA-Seq data from TCGA (n=52) with p-values generated using paired t-tests


Indicated cell lines were transfected with indicated constructs , and treated with veliparib . Cell growt was assessed LNCaP cell growth : Control transfection , p = 0 . 0220 ; BRCA1 transfection , p = 0 . 67787 ; BRCA2 transfection , p = 0 . 4676 . C4 - 2 cell growth : Control transfection , p = 0 . 0354 ; BRCA1 transfection , p = 0 . 1638 ; BRCA2 transfection , p = 0 . 2519 . 22Rv1 cell growth : Control transfection , p = 0 . 0039 ; BRCA1 transfection , p = 0 . 1085 ; BRCA2 transfection , p = 0 .

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Indicated cell lines were transfected with indicated constructs, and treated with veliparib. Cell growt was assessed LNCaP cell growth: Control transfection, p=0.0220; BRCA1 transfection, p=0.67787; BRCA2 transfection, p=0.4676. C4-2 cell growth: Control transfection, p=0.0354; BRCA1 transfection, p=0.1638; BRCA2 transfection, p=0.2519. 22Rv1 cell growth: Control transfection, p=0.0039; BRCA1 transfection, p=0.1085; BRCA2 transfection, p=0.2781


Indicated cell lines were transfected with indicated constructs , and treated with veliparib DDR via γH2AX was assessed LNCaP γH2AX : Control transfection , p = 0 . 0008 ; BRCA1 transfection , p = 0 . 9035 ; BRCA2 transfection , p = 0 . 4685 . C4 - 2 γH2AX : Control transfection , p = 0 . 0009 ; BRCA1 transfection , p = 0 . 6362 ; BRCA2 transfection , p = 0 . 4217 . 22Rv1 γH2AX : Control transfection , p < 0 . 0001 ; BRCA1 transfection , p = 0 . 4698 ; BRCA2 transfection , p = 0 .

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

Indicated cell lines were transfected with indicated constructs, and treated with veliparib DDR via γH2AX was assessed LNCaP γH2AX: Control transfection, p=0.0008; BRCA1 transfection, p=0.9035; BRCA2 transfection, p=0.4685. C4-2 γH2AX: Control transfection, p=0.0009; BRCA1 transfection, p=0.6362; BRCA2 transfection, p=0.4217. 22Rv1 γH2AX: Control transfection, p<0.0001; BRCA1 transfection, p=0.4698; BRCA2 transfection, p=0.4937


A - C . CHO cells expressing PD - L1 alone ( A ) or co - expressing CD80 ( B ) or CD86 ( C ) were stained with CD80 Ig , PD - 1 Ig , CTLA - 4 Ig or anti - PD - L1 Ab clones MIH1 , MIH3 , 29E . 2A3 ) at 1µg / ml for 30 min at 37 ° C . Representative FACS plots show staining plotted against total PD - L1 mCherry levels .

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

A-C. CHO cells expressing PD-L1 alone (A) or co-expressing CD80 (B) or CD86 (C) were stained with CD80 Ig, PD-1 Ig, CTLA-4 Ig or anti-PD-L1 Ab clones MIH1, MIH3, 29E.2A3) at 1µg/ml for 30 min at 37°C. Representative FACS plots show staining plotted against total PD-L1 mCherry levels.


E . Western blot analysis of an immunoprecipitation by anti - mCherry antibody ( PDL1 ) from indicated DG - 75 cell lysates , with and without the BS3 crosslinker , and probed for mCherry ( PD - L1 ) or GFP ( CD80 ) .

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E. Western blot analysis of an immunoprecipitation by anti-mCherry antibody (PDL1) from indicated DG-75 cell lysates, with and without the BS3 crosslinker, and probed for mCherry (PD-L1) or GFP (CD80).


A . Transendocytosis assays were carried out overnight using CHO donor cells ( CD80GFP alone or CD80GFP with PD - L1mCherry co - expression ) incubated with CTLA - 4 recipient cells at a ratio of 1 : 1 . Recipient cells either lacked CTLA - 4 ( No CTLA - 4 ) or expressed CTLA - 4 . FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA - 4 + recipients .

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A. Transendocytosis assays were carried out overnight using CHO donor cells (CD80GFP alone or CD80GFP with PD-L1mCherry co-expression) incubated with CTLA-4 recipient cells at a ratio of 1:1. Recipient cells either lacked CTLA-4 (No CTLA-4) or expressed CTLA-4. FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA-4+ recipients.


C - F . Transendocytosis assays were performed at a ratio of 2 donor : 1 CTLA - 4 recipient for the times indicated . ( C ) , Representative FACS plots showing CD80GFP levels and ( D ) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA - 4 control ( mean ± SEM , 3 independent experiments , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( E ) Representative FACS plots showing PD - L1mCherry level at time points indicated . ( F ) Full kinetic analysis of PD - L1mCherry level on donor cells ( mean ± SEM , 3 independent experiments ) .

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

C-F. Transendocytosis assays were performed at a ratio of 2 donor:1 CTLA-4 recipient for the times indicated. (C), Representative FACS plots showing CD80GFP levels and (D) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA-4 control (mean ± SEM, 3 independent experiments, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test). (E) Representative FACS plots showing PD-L1mCherry level at time points indicated. (F) Full kinetic analysis of PD-L1mCherry level on donor cells (mean ± SEM, 3 independent experiments).


D . Levels of CD86GFP and PD - L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA - 4 WT or Del36 plotted relative to no CTLA - 4 control ( mean ± SEM , 6 independent experiments ) . * * * * P ≤ 0 . 0001 , RM one - way ANOVA .

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D. Levels of CD86GFP and PD-L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA-4 WT or Del36 plotted relative to no CTLA-4 control (mean ± SEM, 6 independent experiments). ****P≤0.0001, RM one-way ANOVA.


A - B . APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was used to detect PD - L1 in DG - 75 cells co - expressing CD80GFP ( A ) or CD86GFP ( B ) . Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA - 4 , CTLA - 4 WT or CTLA - 4 Del36 ( at a ratio of 1 : 1 ) for the indicated durations . ( A ) shows representative FACS plots of CD80GFP vs . PD - 1 Ig at the time points indicated , with full kinetic data plotted below ( mean ± SEM , 3 independent experiments , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( B ) As for ( A ) except using CD86 - PD - L1 expressing cells .

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A-B. APC-labelled PD-1 Ig (0.75µg/ml) was used to detect PD-L1 in DG-75 cells co-expressing CD80GFP (A) or CD86GFP (B). Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA-4, CTLA-4 WT or CTLA-4 Del36 (at a ratio of 1:1) for the indicated durations. (A) shows representative FACS plots of CD80GFP vs. PD-1 Ig at the time points indicated, with full kinetic data plotted below (mean ± SEM, 3 independent experiments, ***P≤0.001, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test). (B) As for (A) except using CD86-PD-L1 expressing cells.


A . Staining of free PD - L1 with APC - conjugated PD - 1 Ig in DG - 75 cells expressing only PD - L1mCherry , using cell lines with increasing ratios of CD80GFP : PD - L1mCherry compared to CD80GFP only . FACS plots are representative of 4 independent experiments .

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A. Staining of free PD-L1 with APC-conjugated PD-1 Ig in DG-75 cells expressing only PD-L1mCherry, using cell lines with increasing ratios of CD80GFP: PD-L1mCherry compared to CD80GFP only. FACS plots are representative of 4 independent experiments.