Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification ( ALI day 3 ) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS ‐ 6 . Deuterosome numbers are decreased and deuterosome size enlarged in Ccno ‐ deficient MCCs as quantified in ( C ) . Deuterosome ‐ dependent centriole biogenesis is severely reduced in Ccno ‐ deficient MCCs as shown by reduced staining for the early centriole marker SAS ‐ 6 .

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Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.


IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno ‐ deficient MCCs at later stages of centriole biogenesis ( ALI day 7 ) . Deuterosome number and size were quantified in ( C ) . IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7 , which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno ‐ deficient MCCs as seen in z ‐ projection .

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IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection.


IFstaining for DEUP1 in ( A , B ) was analysed by Imaris 7 . 7 . 2 software to calculate average deuterosome number of wild ‐ type ( n = 17 ) and Ccno ‐ deficient ( n = 19 ) MCCs at ALI day 3 , and wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 . Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3 , and in wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 .

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IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.


A , B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164 , which marks maturing centrioles . At ALI day 7 , centrioles of wild ‐ type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane . The fewer centrioles of Ccno ‐ deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z ‐ projection . Lack of CEP164 indicates immature centrioles in Ccno ‐ deficient MCCs . ( B ) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7 . 7 . 2 software .

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A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.


C , D Double IFstaining using Centrin ‐ and CP110 ‐ specific antibodies shows an almost complete absence of CP110 from Centrin ‐ positive centrioles in the majority of wild ‐ type MCCs at day 7 of ALI cultures . Most Ccno ‐ deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z ‐ projection . ( D ) CP110 ‐ positive MCCs were counted in wild ‐ type and Ccno ‐ deficient MTEC cultures and the percentages of CP110 ‐ positive MCCs from total MCCs calculated .

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C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.


Luciferase sequestration as an assay for in vitro uptake . ( A ) Luciferase cofractionating with vacuoles is proteinase K protected . In vitro uptake reactions of three times the standard volume were performed ( 60 min , 27 ° C ) in the presence or absence of the ATP - regenerating system . The vacuoles were reisolated , washed , resuspended in 150 mM KCl in PS buffer to a concentration of 0 . 23 mg / ml , and split into aliquots . After addition of 1 % Triton X - 100 or buffer , the mixture was digested with the indicated concentrations of proteinase K ( 15 min , 0 ° C ) . Proteinase treatment was stopped by adding one volume of 0 . 5 mM PMSF in 150 mM KCl in PS buffer . Then , luciferase activity was assayed .

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Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.


( B ) Luciferase can be released from the vacuolar lumen by freeze - thaw treatment . An uptake reaction was performed ( 60 min , 27 ° C ) . The vacuoles were reisolated and washed as described in the Materials and Methods section . The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots . One was frozen at − 80 ° C and thawed again slowly , whereas the other remained on ice . The samples were centrifuged ( 15 min , 145 , 000 g , 4 ° C ) . The supernatants ( Sup ) were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl . Luciferase activity in both fractions was determined . The reaction tubes for this experiment had been coated with BSA ( 0 . 5 mg / ml , 15 min at room temperature ) to reduce unspecific binding of luciferase to the surface .

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(B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.


time course and temperature dependence of uptake . ( A ) Standard uptake reactions were performed in the presence or absence of the ATP - regenerating system . After the indicated times at 27 ° C , the vacuoles were reisolated , washed , treated with protease , and luciferaseuptake activity was assayed .

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time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.


( B ) A standard uptake reaction was performed . After 70 min of incubation , the vacuoles were reisolated , washed , and treated with protease . The vacuoles were reisolated again , resuspended in reaction buffer without luciferase , and incubated for the indicated times at 27 ° C . Aliquots were withdrawn to assay the total luciferase remaining . As a control , luciferase was incubated in reaction buffer for the same periods of time and assayed for activity .

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(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.


Requirements for in vitro uptake . Standard uptake reactions were performed ( 60 min ) ( A ) at different temperatures , or in the presence of different concentrations of ( B ) cytosol , ( C ) ATP , or ( D ) MgCl2 . Luciferase uptake was determined as described in the legend to Fig . 3 A . In B , titration curves for four independent cytosol preparations are shown . The maximal uptake signal of each titration curve was set at 100 % . In D , the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction .

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Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.


In vitro uptake is independent of components involved in homotypic vacuolar fusion . ( A ) Standard uptake reactions were performed ( 60 min , 27 ° C ) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734 - 16 ( Δpep4 ) and DBY5734 - 19 ( Δpho8 ) . Therefore , homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample . Four independent experiments were averaged . Error bars indicate SD . For averaging , uptake and fusion activities of the control sample without inhibitor were set at 100 % . Inhibitors were used at the following concentrations : anti - Sec17p , 1 μM ; anti - Sec18p , 1 μM ; Gdi1p , 2 . 5 μM ; anti - Vam3p , 1 μM ; anti - Vam7p , 1 μM ; and anti - Nyv1p , 1 μM .

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In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM.


( 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 .

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(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 ) .

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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 .

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(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 .

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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 .

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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 .

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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 .

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(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 ) .

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(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 ) .

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(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 .

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

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 .

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

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

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 .

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

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 .

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

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 .

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 .

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

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 B-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

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 .

O O O O B-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

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 .

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 .

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 O O O O O

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 B-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, 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 .

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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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 .

O O O O O B-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

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 B-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

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 B-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

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 .

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

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 .

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

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


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

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

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 .

O O O O O O O O O O B-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

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 .

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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 ) .

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

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 .

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

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

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 B-MEASURED_VAR 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

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

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 B-MEASURED_VAR 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

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 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

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

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 B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 .

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

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

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 .

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

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

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

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 .

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

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

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

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

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

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(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 . (

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(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 .

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

(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 ) .

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

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

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

(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 .

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

(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 .

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(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.