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

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_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

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

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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 ) growth of tumours after castration ( lower panel ) compared to vehicle ( n = 10 ) . PSA was measured every week . Data shown as means ±

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 therefore AR level , with subsequent p - CAMKK2 axis activation in castrated LNCaP xenografts . Bar graph in right panel shows of data from western blot normalized to loading control . Data shown as means ±

O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_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

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 sh2 , compared to control ( Ctr ) . Left , representative immunoblot . Actin is shown as loading control full - blots are shown in Supplementary Figure 21 . Right , amounts of RARα in control knockdown determined by densitometric of immunoblots represented by one shown on left . Values are normalized for actin expressed as multiples of control ( None ) values ; n = 3 .

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 B-MEASURED_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

(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 in control RARα ( - ) maintained in presence or absence of serum for 12 h . Values are expressed as of proteolysis ; n = 3 .

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

(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 ) of degradation due to lysosomes ( c ) macroautophagy ( d ) in 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 of total degradation sensitive to lysosomal inhibitors ; n = 3 . In panels , values are mean ± s . e . m . , differences with control are significant for * 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

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

O O O O O B-MEASURED_VAR O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_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-MEASURED_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

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

O O O O O O O O O O O O O O O O O B-MEASURED_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

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

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

(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 in mouse fibroblasts untreated ( None ) or treated with 40 μM ATRA maintained in presence or absence of serum . Values are expressed as a of proteolysis ; n = 3 .

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

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

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

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


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

O O O O O B-MEASURED_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 B-MEASURED_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 B-MEASURED_VAR O O O O O O

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

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

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

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

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

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

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

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

O O O O O O O O O O O B-MEASURED_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 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 O O O

(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 human RXR receptor , a relevant reporter luciferase plasmid non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units detected in subjected to indicated concentrations of ATRA or three retinoid derivatives for 12 h .

O O O O O O O O O B-MEASURED_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

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

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

O O O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_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

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

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

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

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

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

O O O O O O O B-MEASURED_VAR O O O 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 O O O O O

(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 indicated in homogenates ( Hom ) lysosomes ( Lys ) isolated from untreated ( None ) or treated for 12 h with 20 μM of indicated compounds .

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

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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 ) viability of control ( left ) or LAMP - 2A ( − ) ( right ) fibroblasts exposed to 2 mM or 0 . 5 mM paraquat , respectively , treated with indicated compounds for 12 h before or after 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 indicated concentrations of a plasmid encoding α - synuclein left untreated ( None ) or treated with 1 mM paraquat alone or in presence of 20 μM AR7 ; n = 3 . In b - d , values are mean ± s . e . m . Differences with that are untreated ( marked with asterisk ) or those treated only with paraquat ( § ) were significant for P 0 . 001 . Full - blots are shown in Supplementary Figure 21 .

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(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 same as in d . Top , higher - exposure blot to highlight oligomeric ( oligo ) species . Asterisk denotes nonspecific band . M , monomer ; MW , molecular .

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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 . Armi immunoisolated from OSCs using anti - Armi antibodies contains Yb , SoYb , Vret .

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


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

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


C . Silver of components in Vret lane α - Vret ( left ) contained only 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 western blotting were performed in OSCs upon RNAi treatment ( kd ) . Even without association of Yb , Armi , SoYb , Vret associated . Yb Armi remained bound to each other upon SoYb or Vret depletion . level of SoYb ( input ) decreased upon Vret depletion . Likewise , 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 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 Vret to be localized in cytosol . Yb bodies were detected in OSCs upon Armi depletion , but SoYb Vret localized in cytosol . Armi was detected at Yb bodies upon SoYb or Vret depletion . Yb bodies are indicated by 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 ∆ eTud bound to Armi but ∆ eTud did not . Note that WT Yb 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 ∆ 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 deletion mutants ∆ Hel - C ∆ 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 deletion mutants ∆ Hel - C ∆ 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 ∆ Hel - C bind 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 . of transposon mdg1 Bars error bars represent means ± values of three independent experiments . p values were calculated by t - . * : p < 0 . 05 .

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 presence absence of Hel - C domain in OSCs . D . Mapping of tj - piRNAs produced in presence absence of 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 abundances of transposon - repressing piRNAs non - transposon - repressing piRNAs in presence absence of Hel - C domain . Orange blue dots indicate number of normalized piRNA reads mapped uniquely to transposons in reverse orientation coding genes in 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 . relative enrichments of RNAs coimmunopurified with WT Yb ∆ Hel - C . Bars error bars represent means ± 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 ] , CG9257 [ 34 ] ) are also shown . rp49 was used as a negative control . p value was calculated by t - . * * * : 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 - 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 F129A mutants ( green ) impaired Yb 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 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 - tj - piRNAs produced in flamKG mutant heterozygote ovaries .

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


A of WT , dja6 , dja5 , dja6 dja5 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 chlorophyll of 6 - week - old WT , dja6 dja5 complemented ( B ) on MS medium with 2 % sucrose , scale bars = 0 . 5 cm ; WT , dja6 , dja5 RNAi mutants ( C ) grown on soil , scale bars = 1 cm . Fluorescences in ( B ) ( C ) were measured with FluorCam700MF visualized using a pseudocolor index as indicated on 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 RNAi seedlings . FW , fresh . Data are means ± ( 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 ultrastructure of plastids from WT , dja6 , dja5 , dja6 dja5 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 was extracted from 4 - week - old WT , dja6 , dja5 , dja6 dja5 RNAi seedlings , respectively . WT dja6 dja5 used in ( A ) were grown on MS medium with 2 % sucrose , while WT , dja6 , dja5 RNAi mutants used in ( B ) were grown on soil . 10 μg was loaded , except for detection with anti - SUFE1 anti - SUFA antiserum , for which 25 μg were loaded . Specific bands were identified by immunoblotting by their molecular , are indicated by asterisks . Designations of photosynthetic complexes their diagnostic components are labeled on left right , respectively . Fe - S types of Fe - S are shown in parentheses . Actin served as controls to normalize . For each , three independent biological replicates were performed 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 WT , dja6 dja5 RNAi mutants . Fe concentrations in total leaves ( A B ) total chloroplast ( C ) from 4 - week - old WT , dja6 dja5 RNAi mutants were quantified by inductively coupled plasma mass spectrometry , are shown as means ± ( n = 3 biological replicates ) of μg g - 1 dry ( DW ) . WT RNAi mutants used in ( A C ) were grown on soil , while WT 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 value of WT ( two - sample Student ' s t - ; * , 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 result from quantitative transcriptome profiling in WT dja6 dja5 mutant . Statistical was performed using Student ' s t - . Data on X - axis represent Log2 - of FC ( fold change ) between absolute abundances of transcripts identified from dja6 dja5 against WT . Y - axis represents Log10 of FDR ( false discovery rate ) . DEGs ( differentially expressed genes ) with FDR < 0 . 01 FC > 2 are marked in grey , while iron - related DEGs with FDR < 0 . 01 FC > 2 are marked in red . iron - related DEGs labeled with red dots within black boxes are referred to in text . data are on three biological replicates .

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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 indicated gene constructs , respectively . untransformed protoplasts protoplasts transformed with non - fused EBFP were used as controls . protoplasts were further stained by FeRhoNoxTM - 1 ( red ) to visualize distribution of Fe2 + . Green , EBFP signals ; magenta , chloroplast autofluorescence signals ; yellow , regions where EBFP FeRhoNoxTM - 1 signals merged . speckles which DJA6 - EBFP co - localizes with FeRhoNoxTM - 1 are indicated by 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 interactions of DJA6 with SUFE1 SUFC . Total extracts of protoplasts co - transfected with designated constructs were subjected to immunoprecipitation , then analyzed by immunoblotting with antibodies indicated on right . Input ( left lane ) indicates that 100 μg total was loaded on 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 interactions between truncated forms of DJA6 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 chlorophyll of 6 - week - old WT 35Spro : SynDJA6 - FLAG / dja6 dja5 transgenic 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 . Total leaf extracts ( 100 μg ) from WT 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 PCa ( n = 132 ) CRPC ( n = 148 ) were stained via immunohistochemistry for poly ( ADP - ribose ) ( PAR ) , scored by a clinical pathologist ( T . Parsons ) for ( 0 - 3 ) ( 0 - 3 ) PAR score was generated via equation : ( x 1 ) + ( x 2 ) . PAR scores were compared between 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 then were compared to progression - free survival in 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 after multiplex for γH2AX ( green ) , PAR ( red ) , PARP - 1 ( purple ) , with ( 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 representation ( boxed areas ) . Bottom Left : positive for PAR for entirety of each TMA cohort . Bottom Middle : positive for PARP - 1 . Bottom right : γH2AX for entirety of each TMA cohort . Data was considered after a median cutoff , analyzed for statistical significance using two - Student " s t - for PAR , PARP - 1 , 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 - Spearman correlation between PAR γH2AX ( % positive with a median 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 conditions utilized for transcriptomic analyses ( n = 2 ) of HT - sensitive LNCaP . 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 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 ) 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 conditions utilized for transcriptomic analyses ( n = 2 ) of CRPC C4 - 2 . 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 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 ) 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 ( left ) or CRPC ( right ) were queried against expression of these genes in Grasso et al data set in Oncomine . Benign = grey , PCa = blue , metastases = orange . Boxplot was generated using mean expression of PARPi down - regulated genes in indicated data sets . Statistical significance determined by two - Student " s

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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 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 - Student " s t 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 =

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

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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 , treated with either vehicle control or veliparib for six days . was then harvested from tissues used for qPCR of indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each 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 lines were treated as depicted in Figure 2 , labeled with bromodeoxyuridine ( BrdU ) , harvested at indicated time points 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 of homologous recombination ( HR ) gene expression after 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 . union of these three data sets was used to generate signature . Cut - offs for comparison were a p value < 0 . 05 , fold change of 1 . 5 . Venn diagrams show overlapping non - overlapping genes of both down - ( top ) up - regulated ( bottom ) genes in previously - defined HR deficiency signature , PARPi - responsive

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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 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 - Student " s t 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 were treated as depicted in Figure 2 immunoblotted with indicated antisera . Quantifications shown below

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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 , treated with either vehicle control or veliparib for six days . was then harvested from tissues used for qPCR of indicated transcripts . Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples . Each tissue is depicted by a separate bar color . Statistical analyses were performed by Wilcoxon signed rank

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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. Statistical analyses were performed by Wilcoxon signed rank test


C4 - 2 were treated as depicted in Figure 2 . ChIP was performed using indicated antisera , subsequent was isolated used in qPCR reaction using primers designed to amplify 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 treated with 2 . 5uM veliparib ( Vel . ) or vehicle control ( Veh . ) for 24 hours . were then harvested , lysed , differentially centrifuged as described in material methods section , resulting in a soluble fraction ( Sol . ) ( GAPDH serves as control ) or a - tethered fraction ( Teth . ) ( histone H4 serves as control ) . Immunoblots were performed for 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 of indicated HR pathway genes in PCa vs normal samples . Violin plots represent FPKM normalized obtained from matched normal - 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 lines were transfected with indicated constructs , treated with veliparib . growt was assessed LNCaP growth : Control transfection , p = 0 . 0220 ; BRCA1 transfection , p = 0 . 67787 ; BRCA2 transfection , p = 0 . 4676 . C4 - 2 growth : Control transfection , p = 0 . 0354 ; BRCA1 transfection , p = 0 . 1638 ; BRCA2 transfection , p = 0 . 2519 . 22Rv1 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 lines were transfected with indicated constructs , 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 .

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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 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 plotted against total PD - L1 mCherry .

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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 of an immunoprecipitation by anti - mCherry antibody ( PDL1 ) from indicated DG - 75 lysates , with without BS3 crosslinker , 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 ( CD80GFP alone or CD80GFP with PD - L1mCherry co - expression ) incubated with CTLA - 4 recipient at a of 1 : 1 . Recipient either lacked CTLA - 4 ( No CTLA - 4 ) or expressed CTLA - 4 . FACS plots show CD80GFP ligand loss from donor highlighted in blue quadrants in 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 of 2 donor : 1 CTLA - 4 recipient for times indicated . ( C ) , Representative FACS plots showing CD80GFP ( D ) full kinetic of CD80 or CD86 downregulation on donor quantified as a relative to no CTLA - 4 control ( mean ± , 3 independent experiments , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons ) . ( E ) Representative FACS plots showing PD - L1mCherry level at time points indicated . ( F ) Full kinetic of PD - L1mCherry level on donor ( mean ± , 3 independent experiments ) .

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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 . of CD86GFP PD - L1mCherry remaining on donor over a 24h transendocytosis period with CTLA - 4 WT or Del36 plotted relative to no CTLA - 4 control ( mean ± , 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 co - expressing CD80GFP ( A ) or CD86GFP ( B ) . was carried out following transendocytosis using Jurkat expressing no CTLA - 4 , CTLA - 4 WT or CTLA - 4 Del36 ( at a of 1 : 1 ) for indicated durations . ( A ) shows representative FACS plots of CD80GFP vs . PD - 1 Ig at time points indicated , with full kinetic data plotted below ( mean ± , 3 independent experiments , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 : two - way ANOVA with Tukey ' s multiple comparisons ) . ( B ) As for ( A ) except using CD86 - PD - L1 expressing .

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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 . of free PD - L1 with APC - conjugated PD - 1 Ig in DG - 75 expressing only PD - L1mCherry , using 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.


A . DG - 75 B expressing CD80GFP or CD86GFP ( green ) PD - L1mCherry ( red ) were incubated with Jurkat ( nuclei shown in grey ) expressing CTLA - 4 WT or mutant CTLA - 4 Del36 for indicated durations analysed by confocal microscopy . 2 hours prior to endpoint , APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was added . CD80 / CD86 PD - L1 co - localisation at immune synapse is shown in yellow with PD - 1 Ig binding shown in purple . Scale bar , 10µm .

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A. DG-75 B cells expressing CD80GFP or CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat cells (nuclei shown in grey) expressing CTLA-4 WT or mutant CTLA-4 Del36 for the indicated durations and analysed by confocal microscopy. 2 hours prior to assay endpoint, APC-labelled PD-1 Ig (0.75µg/ml) was added. CD80/CD86 and PD-L1 co-localisation at the immune synapse is shown in yellow with PD-1 Ig binding shown in purple. Scale bar, 10µm.


B . Flow cytometry plots of CD80GFP on DG - 75 B - ( gated on CTFR + ) at 24h with graphical representation of MFI in each condition ( RH graph ) . Jreg used , anti - PD - 1 status is indicated above plot . C . Flow cytometry plots of CD28 indicating contact between CD80 + APC PD - 1 + CD28 + responder Jurkat T - , following CD80 transendocytosis . Graphical representation of CD28 MFI in each condition is shown on right . D . Flow cytometry of CD69 on responder Jurkat T - graphical representation of CD69 MFI in each condition . E . Flow cytometry of CD69 on CD28KO ( PD - 1 + ) responder Jurkat T - graphical representation of CD69 + MFI in each condition . Cartoons on left of B - E illustrate type analysed . Data information : Data is representative of 3 independent experiments showing mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons .

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B. Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg cells used, and the anti-PD-1 status is indicated above the plot. C. Flow cytometry plots of CD28 levels indicating contact between the CD80+ APC and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28 MFI in each condition is shown on the right. D. Flow cytometry of CD69 levels on responder Jurkat T-cells and graphical representation of CD69 MFI in each condition. E. Flow cytometry of CD69 levels on CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.


B . Titration of Abatacept ( left panel ) EC50 values of Abatacept ( right panel ) for CD80 only or PD - L1 / CD80 co - expressing DG - 75 . Data information : Data is representative of 3 independent experiments showing mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : paired t - ( B )

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B. Titration of Abatacept staining (left hand panel) and EC50 values of Abatacept (right hand panel) for CD80 only or PD-L1/CD80 co-expressing DG-75. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B)


D . Comparison of PD - L1 detection using PD - 1 - Ig or anti - PD - L1 antibodies ( MIH3 or 29E . 2A3 clone ) with without prior incubation with 10µg / ml of abatacept , on data from ( C ) . E . Graphs showing PD - L1 detection under conditions used in ( C ) following different Abatacept incubation periods ( 30mins , 3h or 6h ) . Data information : Data is representative of 3 independent experiments showing mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : RM one - way ANOVA ( D ) .

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D. Comparison of PD-L1 detection using PD-1-Ig or anti-PD-L1 antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C). E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept incubation periods (30mins, 3h or 6h). Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: RM one-way ANOVA (D).


A . SDS - PAGE of D391N DIS3L2 - complexes . RNAs were radioactively labeled at 5 ' end DIS3L2 - bound RNAs were detected by autoradiography . negative control was performed with HEK293T - Rex ( Control )

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A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)


B . DIS3L2 CLIP reads possess oligo ( U ) 3 ' termini . Histograms of reads with 3 ' - terminal stretches of identical nucleotides . Only oligo ( U ) tails longer than 4 nucleotides are observed .

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B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed.


C . untemplated uridylation is mostly found in proximity of mature 3 ' ends . heat map shows coverage of various types by uridylated reads . class number of uridylated genes / transcripts for each category are indicated on left . of each mature was scaled to 100 % .

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C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%.


D . List of DIS3L2 - bound uridylated miRNAs with number of uridylated reads identified in three replicate CLIPs . Members of let - 7 family are shown in bold .

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D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold.


F . Secondary structure of pre - hsa - let - 7i , with both 5p 3p mature products highlighted in bold . intramolecular ligation position is indicated in dash between nt 28 61 . It corresponds to cleavage positions of RNaseT1 , which leaves 3 ' - terminal guanosine residues .

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F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues.


G . D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs . Scatter plot of transcript abundances estimated from - seq from overexpressing D391N DIS3L2 control HEK293T - Rex . CLIPed transcripts are marked in .

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G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.


A . Types of uridylated ncRNAs identified by DIS3L2 CLIP - seq .

A. Types of uridylated ncRNAs identified by DIS3L2 CLIP-seq.


B . U + read coverage in downstream regions of mature U12 snRNA , Vault1 7SL ncRNAs . full box is representing mature ncRNA , thin indicates region downstream of mature 3 ' end ( extension ) .

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B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).


C . of DIS3L2 D391N RIP of U12 U5D snRNAs showing number of snRNA clones with mature ( Mat ) , extended ( ext ) or trimmed ( Trim ) 3 ' end . + U is number of clones with untemplated 3 ' oligo ( U ) ends . For more details see Figure S2 . ( D ) An example of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) for RNU5D snRNA .

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C. Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature (Mat), extended (ext) or trimmed (Trim) 3' end. +U is number of clones with untemplated 3' oligo(U) ends. For more details see Figure S2. (D) An example of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) for RNU5D snRNA.


E . U + reads mapped to rDNA locus by using artificial chromosome U13369 . 1 .

E. U+ reads mapped to the rDNA locus by using the artificial chromosome U13369.1.


B . Uridylated tRNA reads overlap mainly with 3 ' trailers , T - loop anticodon loop regions . Heat map showing coverage of uridylated reads on pre - tRNAs . pre - tRNA sub - regions ( as indicated in ( A ) ) are in small letters on x - axis .

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B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.


C . Northern blot of RNAs immunoprecipitated with Flag - tagged WT ( DIS3L2 ) D391N ( MUT ) DIS3L2 , AGO2 DIS3 , respectively . blots were probed with radioactively labeled probes for 5 ' 3 ' ends of mature tRNAVal , respectively .

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C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.


D . Sequencing of MUT DIS3L2 - bound tRNAVal tRNALeu confirmed uridylation status of tRNA fragments identified by CLIP - seq . Genomic for each tRNA is indicated on top with 3 ' trailer region separated by a vertical . Non - templated uridine residues are in red .

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D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.


E . Identification of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) mapping to tRNAPhe ( GAA ) tRNAAsn ( GTT ) genes . To right : schematic representation of selected hybrid reads with corresponding color - code .

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E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.


A . DIS3L2 - bound uridylated mRNA reads map to 5 ' 3 ' UTRs .

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A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs.


B . Most 3 ' UTR U + reads match - loop structure of histone mRNAs . Heat map representation of uridylation position .

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B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position.


C . uridylated aberrant RNAs are stabilized in with nonfunctional DIS3L2 . Small RNAs were modified with 3 ' end linker , linker - specific primer was used for cDNA synthesis . Uridylated RNAs were PCR amplified in a semi - quantitative way with same amount of input used for RT reaction . We used a combination of gene - linker - specific primers , - RT ctrl is RT - PCR control , where no reverse transcriptase was added ; PCR control is a reaction with no cDNA added . graph on right summarizes of uridylated nonuridylated 5 ' fragments of OAT mRNA in HEK293T - Rex ( Ctrl ) overexpressing mutant DIS3L2 ( D391N ) .

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C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N).


D . DIS3L2 - bound uridylated RNAs are degraded by DIS3L2 in vivo . RT - PCR amplification of 5 ' mRfs of OAT RPL12 genes 3 ' extended forms of U12 snRNA ( RNU12 ext ) from HEK293T lines with modified expression of DIS3L2 ( as marked on top ) was performed as described in C . Regions of mature 5S rRNA 7SL were used as loading controls . DIS3L2 KO is a DIS3L2 knock - out . DIS3L2 rescue is DIS3L2 KO with stably integrated mutant ( D391N ) wild type ( WT ) DIS3L2 expressing constructs , respectively . Bands marked with * are unspecific primer - dimer PCR products .

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D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products.


E . Metagene of position of DIS3L2 D391N clipped U + reads peak maximum around mRNA transcription start sites ( TSS ) of ChIP - seq data for RNAP II .

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E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II.


F . U + 5 ' mRfs aberrant forms of U12 snRNA are highly enriched in cytoplasm . RT - PCR detection of 5 ' mRfs of OAT RPL12 aberrant form of U12 snRNA was performed as described in C with isolated from fractions , respectively . RT - PCR amplification of of 5S rRNA was used as a positive control for isolation RT reaction in both fractions . identity uridylation status of OAT U + 5 ' mRfs was confirmed by sequencing ( shown on bottom ) . In red are untemplated nucleotides , in green region corresponding to first intron of OAT pre - mRNA . efficiency of subcellular fractionation was monitored by western blot ( right panel , WB ) . DDX5 is nucleoplasmic , ACO2 is mitochondrial , DIS3L2 TUBa are markers .

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F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBa are cytoplasmic markers.