( B ) Vacuoles and cytosols were prepared from sec18 - 1 and from corresponding wild - type ( Wt ) cells grown and spheroplasted at 23 ° C . The isolated vacuoles and the cytosols were exposed to heat treatment ( 10 min , 37 ° C ) . Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations . Control reactions were performed in the absence of ATP . Five independent experiments were averaged ; the sample with vacuoles and cytosol from wild - type cells was used as the 100 % reference . Error bars indicate SD . Mut , mutant .

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

(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.


In vitro uptake depends on GTP hydrolysis and on the V - ATPase . ( A ) Standard uptake reactions were performed in the presence of different concentrations of GTPγS . Luciferase uptake was assayed as in the legend to Fig . 3 A . Four independent experiments were averaged , using the sample without inhibitor as the 100 % reference . Error bars indicate SD . GTPγS did not influence luciferase activity by itself ( not shown ) .

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

In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).


( B ) Standard uptake reactions were performed in the presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in the absence of ATP .

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

(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.


C ) MXE saturation analysis . Whereas increasing amounts of RNA - seq reads should lead to the confirmation of further MXE candidates , more RNA - seq reads might also result in the rejection of previously validated MXEs . The green curves show the number of validated MXEs in relation to the percentage of total RNA - seq reads used for validation . The orange curves indicate the number of initially ' validated MXEs ' that were rejected with increasing amounts of reads . Grey dashed lines indicate the point of saturation , which is defined as the point where a two - fold increase in reads leads to rejection of less than 1 % of the validated MXEs . Of note , whereas the rejection of validated MXEs saturates with 20 % of the data the amount of novel MXE validations is still rapidly increasing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MXE saturation analysis. Whereas increasing amounts of RNA-seq reads should lead to the confirmation of further MXE candidates, more RNA-seq reads might also result in the rejection of previously validated MXEs. The green curves show the number of validated MXEs in relation to the percentage of total RNA-seq reads used for validation. The orange curves indicate the number of initially 'validated MXEs' that were rejected with increasing amounts of reads. Grey dashed lines indicate the point of saturation, which is defined as the point where a two-fold increase in reads leads to rejection of less than 1% of the validated MXEs. Of note, whereas the rejection of validated MXEs saturates with 20% of the data the amount of novel MXE validations is still rapidly increasing.


E ) Size and distribution of multi - cluster MXEs .

E) Size and distribution of multi-cluster MXEs.


A ) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM . Here , we used the Gini coefficient , which is a measure of the inequality among values of a frequency distribution ( Ceriani & Verme , 2012 ) and has successfully been used to determine tissue - enriched gene sets ( Zhang et al , 2017 ) , to determine highly tissue - specific MXEs ( maximum normalized Gini index of cluster ) and MXEs with a broad tissue expression distribution ( minimum Gini index ) . For each MXE cluster the percent - spliced - in ( PSI ) value of the ubiquitous MXE ( minimum Gini index ) is subtracted from the PSI value of the specific MXE ( maximum Gini index of cluster ) ( delta PSI value ) and scaled between - 1 ( broad tissue distribution ) and 1 ( highly tissue specific ) . Each column represents an MXE pair and each row represents MXE expression in a tissue , cell - type , or at a developmental time point . The bar graph summarizes counts where the specific MXE is 1 . 5 fold more spliced in than the ubiquitous MXE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE.


C ) Heatmap showing the the delta PSI values ( PSI value of the non - SNP - containing MXE subtracted from the PSI value of the SNP - containing MXE ) of MXE clusters containing pathogenic SNPs scaled between - 1 and 1 ( blue = high expression non - SNP - containing MXE , red = high expression SNP - containing MXE ) . Columns represent MXE clusters and rows tissues , cell types , and developmental stages . The column bar graph summarizes counts where the SNP - containing MXE is 1 . 5 fold more expressed than the non - SNP - containing MXE , whereas the row bar graph shows this for each tissue , cell type , and developmental stage .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap showing the the delta PSI values (PSI value of the non-SNP-containing MXE subtracted from the PSI value of the SNP-containing MXE) of MXE clusters containing pathogenic SNPs scaled between -1 and 1 (blue = high expression non-SNP-containing MXE, red = high expression SNP-containing MXE). Columns represent MXE clusters and rows tissues, cell types, and developmental stages. The column bar graph summarizes counts where the SNP-containing MXE is 1.5 fold more expressed than the non-SNP-containing MXE, whereas the row bar graph shows this for each tissue, cell type, and developmental stage.


( A ) Surface plasmon resonance ( SPR ) KAP1 - KRAB binding assay . MBP - KRAB was immobilized on the chip . WT or R311E KAP1 RBCC were flowed over the chip . Data points are shown in dark red or blue ; fits are shown as light red or blue lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Surface plasmon resonance (SPR) KAP1-KRAB binding assay. MBP-KRAB was immobilized on the chip. WT or R311E KAP1 RBCC were flowed over the chip. Data points are shown in dark red or blue; fits are shown as light red or blue lines.


( C ) LINE - 1 reporter repression with single point mutants and a previously described KRAB - binding deficient KAP1 variant containing four mutations in the CC domain ( CCmut ; V293S / K296A / M297A / L300S ) .

O O O O O O O O O O O O O O O O O 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) LINE-1 reporter repression with single point mutants and a previously described KRAB-binding deficient KAP1 variant containing four mutations in the CC domain (CCmut; V293S/K296A/M297A/L300S).


( D ) SVA reporter repression with the same set of mutants as in ( B ) . Data were normalized to KAP1 KO cells transfected with an empty vector ( EV ) .

O O O O O O O O O O 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) SVA reporter repression with the same set of mutants as in (B). Data were normalized to KAP1 KO cells transfected with an empty vector (EV).


A - D SQST ‐ 1 : : GFP is very weakly expressed and diffusely localized in the cytoplasm in wild ‐ type larval intestine , but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae . ( A ) and ( C ) represent DIC images of the animals in ( B ) and ( D ) , respectively .

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A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.


E Compared to wild ‐ type , SQST ‐ 1 : : GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay . SQST ‐ 1 : : GFP levels in bp399 mutants are greatly reduced after 12 h of starvation . 200 young adult animals for each genotype were collected for analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.


H , I SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants are separable from LAAT ‐ 1 : : Cherry ‐ labeled lysosomes under normal conditions , but are delivered to lysosomes upon starvation . Some aggregates are enclosed by lysosomes ( inserts ) . Scale bar : 20 μm .

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

H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.


J - Q RNAi inactivation of let ‐ 363 , rpn ‐ 2 , cogc ‐ 1 , or hgrs ‐ 1 suppresses the accumulation of SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants . ( J ) , ( L ) , ( N ) , and ( P ) : DIC images of the animals in ( K ) , ( M ) , ( O ) , and ( Q ) . Scale bar : 20 μm .

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J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.


R SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are greatly reduced by inactivation of rpt ‐ 3 and rpn ‐ 2 . 200 young adult animals for each genotype were collected for analysis .

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

R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.


A , B SQST ‐ 1 : : GFP aggregates are absent in the rpl ‐ 43 ( bp399 ) ; sma ‐ 3 ( wk20 ) mutant intestine .

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

A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.


C Percentage of the indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : many SQST ‐ 1 : : GFP aggregates in all intestinal cells , as in rpl ‐ 43 ( bp399 ) mutants ; M : fewer SQST ‐ 1 : : GFP aggregates in some but not all intestinal cells ; N : no or very few SQST ‐ 1 : : GFP aggregates in intestinal cells . For each genotype , > 30 animals were examined .

C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.


D SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are dramatically decreased in a Western blot when sma ‐ 3 , lin ‐ 35 , or daf ‐ 2 is simultaneously depleted . Loss of function of daf ‐ 16 partially restores SQST ‐ 1 levels in rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutants .

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D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.


E Endogenous LGG ‐ 1 is elevated in a Western blot in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants .

O O O O O O O O O O O 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 Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.


F mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

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F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


G , H No SQST ‐ 1 : : GFP aggregates are observed in the lin ‐ 35 ( n745 ) ; rpl ‐ 43 ( bp399 ) mutant intestine .

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

G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.


I , J Numerous SQST ‐ 1 : : GFP aggregates are formed in rpl ‐ 43 ( bp399 ) ; lin ‐ 15A ( n767 ) mutants .

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I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.


K Endogenous LGG ‐ 1 levels are increased in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants .

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

K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.


L mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


M , N rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutant larvae contain no intestinal SQST ‐ 1 : : GFP aggregates .

M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.


O , P SQST ‐ 1 : : GFP aggregates are partially restored in daf ‐ 16 ( mu86 ) ; rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) animals .

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

O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.


A , B hsp ‐ 4 : : GFP is very weakly expressed in wild ‐ type animals .

A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.


C Percentage of indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined for each genotype .

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C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.


D , E Phsp ‐ 4 : : GFP expression is much higher in rpt ‐ 3 ( RNAi ) animals .

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

D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.


F , G Upregulation of Phsp ‐ 4 : : GFP expression in rpt ‐ 3 ( RNAi ) animals is suppressed by the xbp ‐ 1 ( zc12 ) mutation .

F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.


H , I SQST ‐ 1 : : GFP aggregates are absent in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

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

H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.


J , K Loss of function of xbp ‐ 1 restores SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

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

J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.


M The increase in bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 mRNA levels in npl ‐ 4 . 1 , rpt ‐ 3 , and pep ‐ 2 RNAi animals is dependent on xbp ‐ 1 . * P 0 . 05 , * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

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M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


A Phsp ‐ 60 : : GFP is weakly expressed in wild ‐ type animals .

A Phsp‐60::GFP is weakly expressed in wild‐type animals.


B egl ‐ 46 ( RNAi ) causes upregulation of Phsp ‐ 60 : : GFP .

B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.


C Percentage of indicated mutant animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined in each group .

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C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.


D Levels of SQST ‐ 1 : : GFP in various genetic mutants . 200 young adult animals for each genotype were collected for analysis .

O O 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 Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.


E , F Loss of function of atfs ‐ 1 suppresses the upregulation of Phsp ‐ 60 : : GFP in egl ‐ 46 ( n1126 ) mutants . ( E ) : DIC image of ( F ) .

O O O O O O O O O O O O O O O O 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 Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).


G , H The dramatic decrease in the number of SQST ‐ 1 : : GFP aggregates in the intestine in rpl ‐ 43 ( bp399 ) ; egl ‐ 46 ( RNAi ) mutants ( G ) is restored by simultaneous loss of function of atfs ‐ 1 ( gk3094 ) ( H ) .

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

G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).


I , J GFP : : LGG ‐ 1 forms numerous puncta in the intestine in egl ‐ 46 ( RNAi ) ( I ) , gfi ‐ 1 ( RNAi ) ( J ) animals .

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

I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.


Representative immunofluorescence images ( left ) of GAPDH ( red ) in HeLa cells untreated ( Utr ) , or treated with IR , etoposide ( ETO ) , adriamycin ( ADR ) , and camptothecin ( CPT ) as indicated . DNA was stained with DAPI ( blue ) . Quantitative analysis ( right ) of nuclear GAPDH . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

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

Representative immunofluorescence images (left) of GAPDH (red) in HeLa cells untreated (Utr), or treated with IR, etoposide (ETO), adriamycin (ADR), and camptothecin (CPT) as indicated. DNA was stained with DAPI (blue). Quantitative analysis (right) of nuclear GAPDH. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


Immunoblot ( left ) and quantification ( right ) of endogenous GAPDH in subcellular fractions of HeLa cells treated 4 Gy IR and recovered for 4 h . β - Tubulin and Lamin A / C served as indicators for the cytoplasmic and nuclear fractions . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

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Immunoblot (left) and quantification (right) of endogenous GAPDH in subcellular fractions of HeLa cells treated 4 Gy IR and recovered for 4 h. β-Tubulin and Lamin A/C served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


Immunoblot ( upper ) and quantification ( lower ) of phospho - Src - Y529 in HeLa cells collected at the indicated time points after 4 Gy IR treatment . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

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Immunoblot (upper) and quantification (lower) of phospho-Src-Y529 in HeLa cells collected at the indicated time points after 4 Gy IR treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


HeLa cells were pretreated with or without 10 μM PP2 for 24 h and exposed to 4 Gy IR . After 4 h of recovery , nuclear and cytoplasmic fractions were separated and subjected to immunoblot with indicated antibodies ( left ) . Quantification of nuclear GAPDH is in the right panel . β - Tubulin and Lamin B1 served as indicators for the cytoplasmic and nuclear fractions . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

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

HeLa cells were pretreated with or without 10 μM PP2 for 24 h and exposed to 4 Gy IR. After 4 h of recovery, nuclear and cytoplasmic fractions were separated and subjected to immunoblot with indicated antibodies (left). Quantification of nuclear GAPDH is in the right panel. β-Tubulin and Lamin B1 served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


E - F . Immunoblot of anti - GAPDH immunoprecipitates from HEK293T treated with 4 Gy IR and recovered for 4 h ( E ) , or with 5 μM ADR for 2 h ( F ) .

O O O O O O 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

E-F. Immunoblot of anti-GAPDH immunoprecipitates from HEK293T treated with 4 Gy IR and recovered for 4 h (E), or with 5 μM ADR for 2 h (F).


Cell viability analysis of HeLa cells stably expressing the sh - NC and sh - GAPDH were treated with the indicated concentration of ADR for 48 h . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Cell viability analysis of HeLa cells stably expressing the sh-NC and sh-GAPDH were treated with the indicated concentration of ADR for 48 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , representative immunofluorescence images of γH2AX ( C , green ) in HeLa cells transfected with scramble siRNA or si - GAPDH after 4 Gy IR exposure and recovered for 4 h . DNA was stained with DAPI ( blue ) . Right quantification of γH2AX foci / cell ( n = 50 ) . Mann - Whitney U - test . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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Left, representative immunofluorescence images of γH2AX (C, green) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right quantification of γH2AX foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , representative immunofluorescence images of 53BP1 ( D , red ) in HeLa cells transfected with scramble siRNA or si - GAPDH after 4 Gy IR exposure and recovered for 4 h . DNA was stained with DAPI ( blue ) . Right , quantification of 53BP1 foci / cell ( n = 50 ) . Mann - Whitney U - test . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Left, representative immunofluorescence images of 53BP1 (D, red) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right, quantification of 53BP1 foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of γH2AX in HeLa cells pre - transfected with scrambled siRNA or si - GAPDH , recovered at indicated points after 4 Gy IR exposure . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (left) and quantification (right) of γH2AX in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at indicated points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of HR proteins in HeLa cells transfected with scrambled siRNA and si - GAPDH . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (left) and quantification (right) of HR proteins in HeLa cells transfected with scrambled siRNA and si-GAPDH. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Quantification of RAD51 ( left ) or γH2AX ( right ) foci / cell in HeLa cells pre - transfected with scrambled siRNA or si - GAPDH , recovered at the indicated time points after 4 Gy IR exposure ( n = 50 ) . Mann - Whitney U - test . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Quantification of RAD51 (left) or γH2AX (right) foci/cell in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at the indicated time points after 4 Gy IR exposure (n=50). Mann-Whitney U-test. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in HeLa cells with or without GAPDH knockdown treated with 50 μg / mL CHX at the indicated time points . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (left) and quantification (right) of RAD51 in HeLa cells with or without GAPDH knockdown treated with 50 μg/mL CHX at the indicated time points. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in GAPDH knockdown HeLa cells and control cells treated with or without MG132 ( 10 μM ) for 10 h . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (left) and quantification (right) of RAD51 in GAPDH knockdown HeLa cells and control cells treated with or without MG132 (10 μM) for 10 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in HeLa cells at different durations of 50 μg / mL CHX administration with or without TSA ( 10 μM , 12 h ) treatment . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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 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

Immunoblot (left) and quantification (right) of RAD51 in HeLa cells at different durations of 50 μg/mL CHX administration with or without TSA (10 μM, 12 h) treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot of cell lysates or anti - RAD51 immunoprecipitates from HeLa cells with scramble siRNA or si - GAPDH transfection treated with MG132 ( 10 μM ) for 2 h .

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Immunoblot of cell lysates or anti-RAD51 immunoprecipitates from HeLa cells with scramble siRNA or si-GAPDH transfection treated with MG132 (10 μM) for 2 h.


Immunoblot ( left ) and quantification ( right ) of Flag - RAD51 in cell lysates from HEK293T cells transfected with indicated Flag - RAD51 mutations . Endogenous RAD51 and Flag - RAD51 are denoted respectively . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (left) and quantification (right) of Flag-RAD51 in cell lysates from HEK293T cells transfected with indicated Flag-RAD51 mutations. Endogenous RAD51 and Flag-RAD51 are denoted respectively. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot of cell lysates or anti - Flag immunoprecipitates from HEK293T cells transfected with indicated Flag - RAD51 mutations and then exposed to 10 μM MG132 . Immunoblot of cell lysates or anti - Flag immunoprecipitates from HEK293T cells transfected with HA - Ubiquitin and indicated Flag - RAD51 mutations and then exposed to 10 μM MG132 .

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Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with indicated Flag-RAD51 mutations and then exposed to 10 μM MG132. Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with HA-Ubiquitin and indicated Flag-RAD51 mutations and then exposed to 10 μM MG132.


HeLa cells were transfected with or without Flag - GAPDH and HA - HDAC1 , then subjected to immunoprecipitation and immunoblots as indicated . Recombinant HDAC1 protein and recombinant GAPDH protein were co - incubated at 4 ℃ for 2 h . In vitro immunoprecipitation was performed using anti - HDAC1 and subjected to immunoblots as indicated .

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

HeLa cells were transfected with or without Flag-GAPDH and HA-HDAC1, then subjected to immunoprecipitation and immunoblots as indicated. Recombinant HDAC1 protein and recombinant GAPDH protein were co-incubated at 4℃ for 2 h. In vitro immunoprecipitation was performed using anti-HDAC1 and subjected to immunoblots as indicated.


HeLa cells were treated with or without 4 Gy IR . The indicated cells were lysed for immunoprecipitation with anti - HDAC1 and immunoblotting analysis ( left ) for anti - Maspin ( I ) Quantitative analysis ( right ) of indicated protein levels . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 .

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

HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-Maspin (I) Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001


HeLa cells were treated with or without 4 Gy IR . The indicated cells were lysed for immunoprecipitation with anti - HDAC1 and immunoblotting analysis ( left ) for anti - GAPDH ( J ) . Quantitative analysis ( right ) of indicated protein levels . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 .

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

HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-GAPDH (J). Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001


Co - immunoprecipitation was performed to determine the interaction between endogenous GAPDH and Maspin , full - length Flag - HDAC1 , and truncated forms of Flag - HDAC1 in HeLa cells , untreated ( L ) or treated with 4 Gy IR ( M ) . Asterisks indicate nonspecific bands .

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

Co-immunoprecipitation was performed to determine the interaction between endogenous GAPDH and Maspin, full-length Flag-HDAC1, and truncated forms of Flag-HDAC1 in HeLa cells, untreated (L) or treated with 4 Gy IR (M). Asterisks indicate nonspecific bands.


Immunoblot ( upper ) and quantification ( lower ) of RAD51 in HEK293T cells transfected with empty vector , Flag - HDAC1 , Flag - HDAC1 - S3 ( ∆ 306 - 353 ) , and Flag - HDAC1 - H141A . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (upper) and quantification (lower) of RAD51 in HEK293T cells transfected with empty vector, Flag-HDAC1, Flag-HDAC1-S3 (∆306-353), and Flag-HDAC1-H141A. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


HeLa cells were transfected with empty vector , Flag - HDAC1 , and Flag - HDAC1 - H141A , then subjected to immunoprecipitation and immunoblots as indicated .

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HeLa cells were transfected with empty vector, Flag-HDAC1, and Flag-HDAC1-H141A, then subjected to immunoprecipitation and immunoblots as indicated.


Left , GAPDH knockdown HeLa cells transfected with Flag - HDAC1 were treated with 4 Gy IR , fixed after 4 h , and immunostained with anti - RAD51 ( red ) . DNA was stained with DAPI ( blue ) . The number of RAD51 foci per cell is shown ( right , n = 50 ) . Mann - Whitney U - test . Scale bars , 5 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Left, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti-RAD51 (red). DNA was stained with DAPI (blue). The number of RAD51 foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( upper ) and quantification ( lower ) of γH2AX in GAPDH knockdown HeLa cells transfected with or without Flag - HDAC1 at the different time points after 4 Gy IR exposure . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

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

Immunoblot (upper) and quantification (lower) of γH2AX in GAPDH knockdown HeLa cells transfected with or without Flag-HDAC1 at the different time points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , GAPDH knockdown HeLa cells transfected with Flag - HDAC1 were treated with 4 Gy IR , fixed after 4 h , and immunostained with anti - γH2AX ( green ) . DNA was stained with DAPI ( blue ) . The number of γH2AX foci per cell is shown ( right , n = 50 ) . Mann - Whitney U - test . Scale bars , 5 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

Left, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti- γH2AX (green). DNA was stained with DAPI (blue). The number of γH2AX foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


b ) The affinity of the UVRAG - C2 domain for phospholipids was assessed using a protein - lipid overlay assay . The left panel indicates the identity of lipid species on PtdIns strips . Bacterial - purified GST fusion of UVRAG - C2 ( wild type , WT ) , but not GST , binds PtdIns ( 3 ) P , PtdIns ( 4 ) P and PtdIns ( 5 ) P . The K78A / R82A mutant of C2 is defective for phospholipid - binding , whereas the K87A / N88A mutant is partially impaired . Binding of the FYVE and PH domains of Hrs and FAPP1 , respectively , to PtdIns ( 3 ) P and PtdIns ( 4 ) P served as quality controls ( right ) . PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; LPA , lysophosphatidic acid ; LPC , lysophosphatidylcholine ; S1P , sphingosine 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

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

b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( c ) The relative affinity of the GST - fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments ( n = 5 ) . Error bars represent standard deviation ( s . d . ) . A . U . , arbitrary units .

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(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.


( d ) Binding of full - length UVRAG ( untagged ) to lipid blots . The same PtdIns strips ( in b ) were used and immunoblotted with antibodies against IgG ( right ) and UVRAG ( left ) .

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(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).


( g ) Pulldown of UVRAG - C2 domain GST - fusion proteins by liposomes bearing the indicated phosphoinositides . GST - Hrs - FYVE and GST - FAPP1 - PH are also shown .

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(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.


( a - k ) Confocal analyses of the subcellular co - localization of endogenous UVRAG with ER markers , including endogenous calnexin ( red , a ) , endogenous PDI ( red , b ) , and overexpressed DSred - ER ( red , c ) ; with Golgi markers , including endogenous GM130 and p115 ( red , d , e , cis - Golgi ) , overexpressed GFP - Man II ( green , f , medial - Golgi ) , endogenous TGN46 ( red , g , trans - Golgi ) , and overexpressed PtdIns ( 4 ) P probe , GFP - FAPP1 - PH ( green , h ) ; with endogenous coatomer proteins , including Sec31 ( red , i , COPII - related ) and β ′ - COP ( red , j , COPI - related ) ; and with the endosome marker GFP - p40phox ( PX ) ( green , k ) in HeLa cells . The insets show a high magnification of the selected areas . Scale bars , 10 μm . ( l ) Confocal microscopic quantification of co - localization of UVRAG with the indicated markers ( data are mean ± s . d . , n = 150 cells obtained by gathering data from three independent experiments ) ; * * P0 . 01 ; * * * P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.


a ) UVRAG is released from the ER membrane in cells overexpressing MTMR3 . HeLa cells transfected with MTMR3 - mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti - UVRAG antibody and processed for confocal microscopy . Asterisks indicate MTMR3 - transfected cells ( left ) . The percentage of transfected cells with diffused staining of UVRAG was quantified ( middle ) . Data are mean ± s . d . ; n = 240 cells collected from four independent experiments ; * * P0 . 01 . Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3-mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.; n = 240 cells collected from four independent experiments; **P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right).


( b ) Wortmannin treatment releases UVRAG from the ER . HeLa cells were treated with 100 nM wortmannin for 1h , and processed for confocal microscopy using anti - UVRAG and anti - PDI antibodies ( top panel ) . UT , untreated . Insets highlight the relative localization of UVRAG at the ER . Confocal co - localization is indicated ( bottom left panel ) . Data are mean ± s . d . ; n = 75 cells for each group from three independent experiments ; * * * P0 . 001 . Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting ( bottom right panel ) .

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

(b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel).


( c ) Mutation of the C2 domain leaves UVRAG unable to associate with the ER . HeLa cells expressing Flag - UVRAG ( WT ) or its K78A / R82A mutant were fixed and processed for confocal microscopy for co - localization with the ER marker PDI ( top panel ) . Insets highlight the relative distribution of UVRAG at the ER . UVRAG and endogenous PDI expression were confirmed by immunoblotting ( bottom panel ) . See Supplementary Fig . S9 for uncropped data . Scale bars , 10 μm .

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(c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag-UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.


( a ) The UVRAG C - terminal 270 - 442 region interacts with RINT - 1 . Cells ( HEK293T ) were co - transfected with HA - RINT - 1 , together with Flag - UVRAG or its mutant derivatives , and whole - cell lysates ( WCLs ) were immunoprecipitated ( IP ) with anti - Flag , followed by immunoblotting ( IB ) with an anti - HA antibody .

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(a) The UVRAG C-terminal 270-442 region interacts with RINT-1. Cells (HEK293T) were co-transfected with HA-RINT-1, together with Flag-UVRAG or its mutant derivatives, and whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with an anti-HA antibody.


c ) Interaction between endogenous UVRAG and RINT - 1 . WCLs of HEK293T cells were used for immunoprecipitation with control serum ( control ) or an anti - UVRAG ( left ) or anti - RINT - 1 antibody ( right ) , followed by immunoblotting with the indicated antibodies . The bottom panel shows endogenous protein expression .

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c) Interaction between endogenous UVRAG and RINT-1. WCLs of HEK293T cells were used for immunoprecipitation with control serum (control) or an anti-UVRAG (left) or anti-RINT-1 antibody (right), followed by immunoblotting with the indicated antibodies. The bottom panel shows endogenous protein expression.


d ) ER localization of RINT - 1 , ZW10 and UVRAG . HeLa cells were stained with anti - UVRAG ( green ) , anti - PDI ( ER marker , red ) , and anti - ZW10 or anti - RINT - 1 ( blue ) , followed by confocal microscopy . Insets highlight UVRAG co - localization with RINT - 1 and ZW10 at the ER . Scale bars , 10 μm .

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d) ER localization of RINT-1, ZW10 and UVRAG. HeLa cells were stained with anti-UVRAG (green), anti-PDI (ER marker, red), and anti-ZW10 or anti-RINT-1 (blue), followed by confocal microscopy. Insets highlight UVRAG co-localization with RINT-1 and ZW10 at the ER. Scale bars, 10 μm.


( e ) Interaction between endogenous UVRAG and the RINT - 1 - ZW10 - NAG tethering complex proteins . WCLs of HEK293T cells were used for immunoprecipitation with control serum ( IgG ) or an anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) , anti - ZW10 ( third panel ) and anti - NAG ( fourth panel ) , followed by immunoblotting with the indicated antibodies . As input , 10 % of the WCLs was used . Syn 18 , syntaxin 18 ; Syn 6 , syntaxin 6 . See Supplementary Fig . S9 for uncropped data .

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(e) Interaction between endogenous UVRAG and the RINT-1-ZW10-NAG tethering complex proteins. WCLs of HEK293T cells were used for immunoprecipitation with control serum (IgG) or an anti-UVRAG (first panel), anti-RINT-1 (second panel), anti-ZW10 (third panel) and anti-NAG (fourth panel), followed by immunoblotting with the indicated antibodies. As input, 10% of the WCLs was used. Syn 18, syntaxin 18; Syn 6, syntaxin 6. See Supplementary Fig. S9 for uncropped data.


( a ) UVRAG knockdown impairs the interaction of the RINT - 1 - ZW10 complex with COPI in an autophagy - independent manner . Cells ( HEK293T ) were transfected with control shRNA , UVRAG shRNA or Atg16L1 shRNA for 72 h . Whole - cell lysates ( WCLs ) were used for immunoprecipitation with anti - RINT - 1 ( first and third panels ) , anti - ZW10 ( second panel ) , or anti - UVRAG ( fourth panel ) , followed by immunoblotting with the indicated antibodies . Input represents 10 % whole - cell lysates . Note that β ′ - COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown . See Supplementary Fig . S9 for uncropped data . (

(a) UVRAG knockdown impairs the interaction of the RINT-1-ZW10 complex with COPI in an autophagy-independent manner. Cells (HEK293T) were transfected with control shRNA, UVRAG shRNA or Atg16L1 shRNA for 72 h. Whole-cell lysates (WCLs) were used for immunoprecipitation with anti-RINT-1 (first and third panels), anti-ZW10 (second panel), or anti-UVRAG (fourth panel), followed by immunoblotting with the indicated antibodies. Input represents 10% whole-cell lysates. Note that β′-COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown. See Supplementary Fig. S9 for uncropped data. (


( b ) Retrograde transport of tsO45 - VSVG - KDELR - YFP from the Golgi to the ER is inhibited by UVRAG knockdown . HeLa cells serially transfected with UVRAG shRNA or control shRNA , then with VSVG - KDELR - YFP expression vector , were incubated at 32 ° C ( permissive condition ) overnight . On shifting to 40 ° C ( non - permissive condition ) , the ER - like distribution pattern of VSVG - KDELR - YFP was registered . Representative confocal images of the distribution pattern of VSVG - KDELR at 40 ° C and its co - localization with PDI are shown ( upper panel ) . Data are mean ± s . d . , n = 100 cells obtained from three independent experiments ; * * P0 . 01 ( bottom panel ) . Scale bars , 10 μm .

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(b) Retrograde transport of tsO45-VSVG-KDELR-YFP from the Golgi to the ER is inhibited by UVRAG knockdown. HeLa cells serially transfected with UVRAG shRNA or control shRNA, then with VSVG-KDELR-YFP expression vector, were incubated at 32°C (permissive condition) overnight. On shifting to 40°C (non-permissive condition), the ER-like distribution pattern of VSVG-KDELR-YFP was registered. Representative confocal images of the distribution pattern of VSVG-KDELR at 40°C and its co-localization with PDI are shown (upper panel). Data are mean±s.d., n = 100 cells obtained from three independent experiments; **P0.01 (bottom panel). Scale bars, 10 μm.


( c ) UVRAG interaction with bothPtdIns ( 3 ) P and RINT - 1 is required for COPI - dependent retrograde transport , as shown by the redistribution of VSVG - KDELR from the Golgi to the ER . HeLa cells were transfected with control shRNA or UVRAG shRNA . The UVRAG - depleted cells were then complemented with empty vector , Flag - UVRAG , UVRAGΔ270 − 442 , UVRAGK78A / R82A or UVRAGΔC2 , along with the transfection of VSVG - KDELR - YFP . The ER pattern for the chimaeric KDELR was quantified ( left panel ) . Data are the mean ± s . d . ; n = 200 cells obtained from three independent experiments ; * P0 . 05 ; * * P0 . 01 . Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting ( right panel ) .

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(c) UVRAG interaction with bothPtdIns(3)P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG-KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag-UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG-KDELR-YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.; n = 200 cells obtained from three independent experiments; *P0.05; **P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel).


( d ) PtdIns ( 3 ) Pdepletion inhibits Golgi - to - ER retrograde transport of VSVG - KDELR . HeLa cells were transfected with empty vector , wild - type MTMR3 , the C413S mutant of MTMR3 , or treated with wortmannin or rapamycin , and then the ER pattern of VSVG - KDELR was quantified . UT , untreated . Data are the mean ± s . d . ; n = 100 cells for each group obtained from three independent experiments ; * * P0 . 01 .

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(d) PtdIns(3)Pdepletion inhibits Golgi-to-ER retrograde transport of VSVG-KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG-KDELR was quantified. UT, untreated. Data are the mean±s.d.; n = 100 cells for each group obtained from three independent experiments; **P0.01.


e ) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment . WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti - UVRAG or anti - RINT - 1 antibody , followed by immunoblotting with the indicated antibodies . The input panel shows endogenous protein expression . See Supplementary Fig . S9 for uncropped data .

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 O O O O O O O

e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.


( a ) Golgi morphology on UVRAG depletion . HeLa cells were transfected with control or UVRAG - specific shRNA for 72 h and subjected to electron microscopy analysis ( left panel ) . When compared with control cells ( left ) , the Golgi structure was swollen and fragmented in cells lacking UVRAG . The level of Golgi fragmentation was quantified ( right panel ) . Data represent mean ± s . d . ; n = 50 cells obtained by gathering data from two independent experiments ; * P0 . 05 . Scale bars , 500 nm .

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(a) Golgi morphology on UVRAG depletion. HeLa cells were transfected with control or UVRAG-specific shRNA for 72 h and subjected to electron microscopy analysis (left panel). When compared with control cells (left), the Golgi structure was swollen and fragmented in cells lacking UVRAG. The level of Golgi fragmentation was quantified (right panel). Data represent mean±s.d.; n = 50 cells obtained by gathering data from two independent experiments; *P0.05. Scale bars, 500 nm.


( b ) Depletion of UVRAG leads to cis - Golgi dispersion . HeLa cells were transfected with control shRNA or UVRAG - specific shRNA expressing GFP as an expression marker and then stained for GM130 ( left panel ; red ; highlighted by arrows ) . Nuclei were stained with DAPI ( blue ) . The dispersed distribution of GM130 in shRNA - transfected cells was quantified ( middle panel ) . Data are the mean ± s . d . ; n = 500 cells obtained from five independent experiments . The expression of UVRAG and GM130 in treated cells is also shown ( right panel ) . Scale bars , 10 μm .

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(b) Depletion of UVRAG leads to cis-Golgi dispersion. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA expressing GFP as an expression marker and then stained for GM130 (left panel; red; highlighted by arrows). Nuclei were stained with DAPI (blue). The dispersed distribution of GM130 in shRNA-transfected cells was quantified (middle panel). Data are the mean±s.d.; n = 500 cells obtained from five independent experiments. The expression of UVRAG and GM130 in treated cells is also shown (right panel). Scale bars, 10 μm.


( c ) The effect of UVRAG on membrane trafficking from the ER to the Golgi . HeLa cells serially transfected with UVRAG shRNA RINT - 1 shRNA or control shRNA , then with VSVG - GFP expression vector , were incubated at 40 ° C ( non - permissive condition ) overnight . The cells were then shifted to 32 ° C ( permissive condition ) for 30 min to allow cargo transit from the ER to the Golgi . Before and after the temperature shift , the cells were fixed and labelled with anti - PDI and anti - GM130 antibodies to define the ER and Golgi region , respectively . Representative confocal microscopy images of the distribution pattern of the retrograde cargo at 40 ° C and 32 ° C are shown ( left panel ) . The Golgi - like distribution pattern of VSVG - GFP was registered ( right panel ) . Data are mean ± s . d . ; n = 100 cells from three independent experiments ; * * P0 . 01 . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of UVRAG on membrane trafficking from the ER to the Golgi. HeLa cells serially transfected with UVRAG shRNA RINT-1 shRNA or control shRNA, then with VSVG-GFP expression vector, were incubated at 40°C (non-permissive condition) overnight. The cells were then shifted to 32°C (permissive condition) for 30 min to allow cargo transit from the ER to the Golgi. Before and after the temperature shift, the cells were fixed and labelled with anti-PDI and anti-GM130 antibodies to define the ER and Golgi region, respectively. Representative confocal microscopy images of the distribution pattern of the retrograde cargo at 40°C and 32°C are shown (left panel). The Golgi-like distribution pattern of VSVG-GFP was registered (right panel). Data are mean±s.d.; n = 100 cells from three independent experiments; **P0.01. Scale bars, 10 μm.


( d , e ) UVRAG interaction with PtdIns ( 3 ) P and RINT - 1 is required or Golgi integrity . ( d ) HeLa cells were transfected with control shRNA ( first row ) or UVRAG shRNA ( second to sixth rows ) . The UVRAG - depleted cells , as indicated by Flag expression , were then transfected with empty vector ( second row ) , UVRAGK78A / R82A - UVRAG ( third row ) , UVRAGΔ270 − 442mutant ( fourth row ) , UVRAGK78A / R82A mutant ( fifth row ) , or UVRAGΔCCD mutant ( sixth row ) , followed by confocal microscopy using anti - GM130 ( red ) for cis - Golgi staining , anti - Flag ( purple ) for the ectopically expressed UVRAG protein expression , and UVRAG for nuclei ( blue ) . Asterisks indicate shRNA - transfected cells and arrows denote the cells reconstituted with GM130 ( wild - type or mutant ) expression . ( e ) The dispersed distribution of GM130 was quantified , and data represent the mean ± s . d . ; n = 200 cells from four independent experiments ; * P0 . 05 ; * * P0 . 01 .

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(d,e) UVRAG interaction with PtdIns(3)P and RINT-1 is required or Golgi integrity. (d) HeLa cells were transfected with control shRNA (first row) or UVRAG shRNA (second to sixth rows). The UVRAG-depleted cells, as indicated by Flag expression, were then transfected with empty vector (second row), UVRAGK78A/R82A-UVRAG (third row), UVRAGΔ270−442mutant (fourth row), UVRAGK78A/R82A mutant (fifth row), or UVRAGΔCCD mutant (sixth row), followed by confocal microscopy using anti-GM130 (red) for cis-Golgi staining, anti-Flag (purple) for the ectopically expressed UVRAG protein expression, and UVRAG for nuclei (blue). Asterisks indicate shRNA-transfected cells and arrows denote the cells reconstituted with GM130 (wild-type or mutant) expression. (e) The dispersed distribution of GM130 was quantified, and data represent the mean±s.d.; n = 200 cells from four independent experiments; *P0.05; **P0.01.


( a , b ) Differential interaction of UVRAG with the RINT - 1 - ZW10 - NAG tethering complex and the beclin - 1 - Bif - 1 - PI ( 3 ) KC3 autophagy complex after treatment with rapamycin ( a ) and HBSS ( b ) . Cells ( HEK293T ) treated with rapamycin ( 50 nM ) in a or with HBSS in b were subjected to immunoprecipitation with anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) or anti - PI ( 3 ) KC3 ( third panel ) , followed by immunoblotting with the indicated antibodies . Input ( 10 % ) shows endogenous protein expression . See Supplementary Fig . S9 for uncropped data .

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

(a,b) Differential interaction of UVRAG with the RINT-1-ZW10-NAG tethering complex and the beclin-1-Bif-1-PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.


( c ) Gel filtration analysis of UVRAG complex formation under normal conditions ( untreated , UT ) and after rapamycin - induced autophagy ( 50 nM ) . Affinity - purified UVRAG complexes from HCT116 cells stably expressing Flag - UVRAG were fractionated by Superose - 6 gel filtration column and the eluates were analysed by western blotting for UVRAG , RINT - 1 , ZW10 , beclin 1 , PI ( 3 ) KC3 and Bif - 1 proteins . Whole - cell lysates ( 2 . 5 % ) were used as the input ( lane - 1 ) . The elution profile of each protein was quantified by densitometry analysis and normalized . Relative densitometry units were plotted against fraction number . Black arrows indicate the positions of the molecular weight size markers . Red arrows indicate the peak shift of the UVRAG eluates .

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(c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag-UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.


( a - c ) UVRAG interaction with beclin 1 and PtdIns ( 3 ) P , but not with RINT - 1 , is required for autophagy - induced Atg9 dispersal . ( a ) HeLa cells were stably transfected with control shRNA ( first row ) or UVRAG shRNA ( second - sixth rows ) . The UVRAG - depleted cells were then transfected with empty vector ( second row ) , Flag - UVRAG ( third row ) , UVRAGΔ270 − 442 mutant ( fourth row ) , UVRAGK78A / R82A mutant ( fifth row ) or UVRAGΔCCD mutant ( sixth row ) , and incubated in complete medium ( untreated , UT , first panel ) , starvation medium ( HBSS 2 h , third panel ) , or treated with rapamycin ( 50 nM 2 h , second panel ) or SMER28 ( 50 μM , 2 h , fourth panel ) . The distribution pattern of endogenous Atg9 ( green ) in the UVRAG ( wild - type or mutant ) - transduced cells ( red ) was analysed by confocal microscopy ( a ) . Asterisks denote condensed ( non - dispersed ) Atg9 in UVRAG - depleted HeLa cells and arrows denote the cells complemented with wild - type or mutant UVRAG .

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(a-c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.


( b , c ) The percentage of cells with Atg9 translocation was quantied ( c ) and the expression of UVRAG and actin was determined by immunoblotting ( b ) . Data represent the means : d : ; n D300 cells from three independent experiments ; NS , not signicant ; P < 0 : 01 ; P < 0 : 001 . Scale bars , 10 m .

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(b,c) The percentage of cells with Atg9 translocation was quantied (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the means:d:; n D300 cells from three independent experiments; NS, not signicant; P <0:01; P <0:001. Scale bars, 10 m.


( d ) Effect of knockdown of UVRAG , beclin 1 andRINT - 1 on the translocation of Atg9 to the LC3 - labelled autophagsomes during autophagy . Control - , UVRAG - , BECN1 - or RINT - 1 - knockdown HeLa cells were incubated in complete medium ( normal condition ) , starvation medium ( HBSS , 2 h ) , or treated with rapamycin ( 100 nM , 2 h ) or SMER28 ( 50 μM , 2 h ) . The cells were then stained for endogenous Atg9 ( green ) and LC3 ( red ) followed by confocal microscopy analysis ( top and bottom left panels ) . The percentage of Atg9 vesicles co - localized with LC3 + - autophagic puncta was quantified ( bottom middle panel ) . Endogenous UVRAG , beclin 1 and RINT - 1 protein expression are shown ( bottom right ) . Data represent mean ± s . d . ; n = 200 cells obtained from three independent experiments . NS , not significant ; * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . Scale bars , 10 μm .

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(d) Effect of knockdown of UVRAG, beclin 1 andRINT-1 on the translocation of Atg9 to the LC3-labelled autophagsomes during autophagy. Control-, UVRAG-, BECN1- or RINT-1-knockdown HeLa cells were incubated in complete medium (normal condition), starvation medium (HBSS, 2 h), or treated with rapamycin (100 nM, 2 h) or SMER28 (50 μM, 2 h). The cells were then stained for endogenous Atg9 (green) and LC3 (red) followed by confocal microscopy analysis (top and bottom left panels). The percentage of Atg9 vesicles co-localized with LC3+-autophagic puncta was quantified (bottom middle panel). Endogenous UVRAG, beclin 1 and RINT-1 protein expression are shown (bottom right). Data represent mean±s.d.; n = 200 cells obtained from three independent experiments. NS, not significant; *P0.05; **P0.01; ***P0.001. Scale bars, 10 μm.


A . Viability analysis of GICs treated with the α - AR antagonists prazosin , ARC 239 , doxazosin , BMY 7378 , and terazosin . GICs were treated with the antagonists or corresponding vehicles for 72 h , and viability was assayed using WST - 1 . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - test .

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A. Viability analysis of GICs treated with the α-AR antagonists prazosin, ARC 239, doxazosin, BMY 7378, and terazosin. GICs were treated with the antagonists or corresponding vehicles for 72 h, and viability was assayed using WST-1. *P<0.05, n=4, two-sided Mann-Whitney U-test.


B . Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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B. Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Viability analysis of patient - derived GICs ( TG1 , TG16 , GBM5 , GBM44 ) and NSCs ( NSC24 , NSC25 , NSC5031 , NSC8853 ) treated with prazosin for 72 h . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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C. Viability analysis of patient-derived GICs (TG1, TG16, GBM5, GBM44) and NSCs (NSC24, NSC25, NSC5031, NSC8853) treated with prazosin for 72 h. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Analysis of the sphere - forming capabilities of GICs using the extreme limiting dilution assay . Cells were seeded in presence of vehicle or 30 µM prazosin ( PRZ ) . Sphere formation was scored 10 days post - seeding . Frequency of sphere - forming cells : Control = 1 / 3 . 88 ( lower 8 . 61 , upper 1 . 95 ) ; prazosin 1 / 248 ( lower 1003 , upper 62 ) , n = 12 , p = 1 . 13 10 - 10 . Overall test for difference in stem cell number between groups .

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D. Analysis of the sphere-forming capabilities of GICs using the extreme limiting dilution assay. Cells were seeded in presence of vehicle or 30 µM prazosin (PRZ). Sphere formation was scored 10 days post-seeding. Frequency of sphere-forming cells: Control=1/3.88 (lower 8.61, upper 1.95); prazosin 1/248 (lower 1003, upper 62), n=12, p= 1.13 10-10. Overall test for difference in stem cell number between groups.


E . Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR , and the neural stem cell markers CD15 and CD133 . Prazosin inhibits cell viability regardless of CD133 or CD15 expression . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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E. Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR, and the neural stem cell markers CD15 and CD133. Prazosin inhibits cell viability regardless of CD133 or CD15 expression. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Imunocytochemical staining of NSCs and GICs cultured in media favoring neuronal ( ß3 - tubulin ) , astroglial ( GFAP ) or oligodendroglial ( O4 ) differentiation ( Diff media ) . ß3 - tub : ß3 - tubulin .

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F. Imunocytochemical staining of NSCs and GICs cultured in media favoring neuronal (ß3-tubulin), astroglial (GFAP) or oligodendroglial (O4) differentiation (Diff media). ß3-tub: ß3-tubulin.


G . Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h . Diff : differentiation . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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G. Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h. Diff: differentiation. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


B , C . In vivo effect of prazosin treatment ( 1 . 5mg / Kg ) on glioblastoma growth . Tumors were initiated with GBM44 GICs ( B ) or GMB5 GICs ( C ) . Left panels : Bioluminescent in vivo images of tumors in mice treated with prazosin ( PRZ ) or vehicle for 45 days . Middle panels : Quantification of the bioluminescent signals . Fold change in total flux represents the ratio : total flux after treatment / total flux before treatment . * P = 0 . 0002 and * P = 0 . 003 for GBM44 and GBM5 respectively , n = 8 , two - sided Mann - Whitney U - test . Right panels : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle , log - rank Mantel - Cox test . The treatment period is shaded in gray .

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B, C. In vivo effect of prazosin treatment (1.5mg/Kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P=0.0002 and *P=0.003 for GBM44 and GBM5 respectively, n=8, two-sided Mann-Whitney U-test. Right panels: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Example of hematoxilin / eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin ( PRZ ) or vehicle . i : tumor infiltration . n : tumor necrosis . Scale bar = 2 mm . GBM44 GICs .

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D. Example of hematoxilin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar = 2 mm. GBM44 GICs.


E . Left panel : Representative flow cytometry plots depicting the percentage of CD133 + glioblastoma cells ( GFP + ) isolated from mice treated with prazosin ( PRZ ) or vehicle . Tumors were initiated with GBM44 GICs . Right panel : Quantification of flow cytometry analyses of CD133 expression by GFP + - tumor cells isolated from xenografts of three prazosin - treated ( PRZ ) mice and three vehicle - treated mice . * P = 0 . 0003 , n = 6 , two - sided Mann - Whitney U - test .

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E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P=0.0003, n=6, two-sided Mann-Whitney U-test.


F . Secondary grafting of tumor cells ( GBM44 - GFP + ) isolated from vehicle or prazosin - treated mice bearing primary tumors . Mice injected with glioblastoma cells isolated from prazosin - treated mice developed tumors at a lower frequency ( 50 % versus 100 % ) . Moreover , mice injected with glioblastoma cells isolated from prazosin - treated mice presented a statistically significant survival benefit . Left Panel : Bioluminescent in vivo images of secondary grafts . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0004 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle - and prazosin - treated mice .

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F. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice.


G . Inhibitory effect of low doses of prazosin ( 0 . 15 mg / Kg ) on glioblastoma growth in vivo . Left panel : Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin ( PRZ ) for 45 days . Tumors were initiated with GBM44 GICs ( compare with panel B ) . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin , log - rank Mantel - Cox test . The treatment period is shaded in gray .

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G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.


A . Viability analysis of GL261 treated with prazosin in vitro . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma . Left panel : Bioluminescent in vivo images of tumors in C57 / Bl6mice treated with vehicle or prazosin ( PRZ ) for 14 days . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0002 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle . log - rank Mantel - Cox test . The treatment period is shaded in gray .

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C. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Hematoxilin / eosin staining of C57 / Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin . Scale bar = 2 mm .

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D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm.


E . Fluorescence imaging of GL261 intra - striatal grafts at 1 and 2 h after an intra - peritoneal injection of BODIPY FL prazosin , the green - fluorescent derivative of prazosin . Scale bar = 2 mm .

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E. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.


A . Immunoblotting for pro - caspase - 3 ( pro - CASP3 ) and active caspase - 3 ( CASP3 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin activates caspase - 3 . ZVAD , a caspase inhibitor , prevents prazosin - induced caspase - 3 activation . kDa : kilodaltons .

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A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.


B . Immunoblotting for pro - caspase - 9 ( pro - CASP9 ) and active caspase - 9 ( CASP9 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin does not activate caspase - 9 .

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B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.


C . Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD , a caspase inhibitor . ZVAD counteracts prazosin - induced GIC death . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Prazosin induces glioblastoma cell apoptosis in vivo . GFP + and GFP - cells were analyzed from tumors in vehicle or prazosin - treated mice . Annexin V / DAPI staining was used to identify apoptotic cells by FACS . Tumors were initiated by GBM44 GICs implantation .

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D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.


E . Dose - response curve of prazosin on GIC survival ( 24h treatment ) . EC50 was determined with curve fit using nonlinear regression .

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E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.


F . Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline , a subtype agonist of the α - ARs . Cirazoline did not alter GIC survival and counteracted only poorly prazosin - induced GIC death . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - test .

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F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.