( h ) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3 , GEMIN4 and ATG5 . Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells .

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(h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells.


( i ) Western blot analysis with anti - UB monoclonal antibody ( mono - and polyubiquitylated proteins , FK2 clone ) in Flag immunoprecipitates performed under stringent denaturing conditions . Flag - AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet - inducible Flag - AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs .

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

(i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs.


( j ) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h . TUBA , α - tubulin loading control .

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

(j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA, α-tubulin loading control.


( k ) Top , RT - qPCR analysis of miR - 16 or let - 7a levels in AGO2 immunoprecipitates from DMSO - and BAF - treated HeLa cells . Error bars represent s . e . m . Bottom , a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

(k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Northern blot of miR - 16 and let - 7a in HeLa cells treated with siRNA for 84 h . Levels of U6 , ethidium - bromide - stained total RNA on northern membrane ( loading ) and 28S and 18S ribosomal RNA on 1 % agarose gel are shown as controls for equal loading .

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

(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading.


( b - d ) RT - qPCR analysis of let - 7a and miR - 16 ( b ) , pre - let - 7a and pre - miR - 16 ( c ) and let - 7a * and miR - 16 * ( d ) in HeLa cells depleted of NDP52 or ATG5 with siRNA ( 84 h ) . n = 3 independent experiments ; error bars represent s . e . m .

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

(b-d) RT-qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h). n = 3 independent experiments; error bars represent s.e.m.


( e ) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let - 7a - let - 7a * labelled on the 3 ′ end of either let - 7 or let - 7a * with FITC . Cells were treated 16 h before with DMSO ( control ) , BAF ( 200 nm ) or geldanamycin ( 10 μM ) . Representative results of one experiment are shown .

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

(e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown.


( f ) Western blot analysis of AGO2 immunoprecipitates ( IP ) used in e .

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(f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e.


( g ) Normalized results of four independent experiments ( n = 4 ) identical to e . Error bars represent s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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(g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


( a ) Renilla luciferase expression controlled by an exogenous siRNA - siRNA * duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA ( black and grey bars correspond to two independent siRNAs ) . Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA - siRNA * duplex . n = 2 independent experiments .

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

(a) Renilla luciferase expression controlled by an exogenous siRNA-siRNA* duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA (black and grey bars correspond to two independent siRNAs). Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA-siRNA* duplex. n = 2 independent experiments.


( b ) Renilla luciferase expression controlled by let - 7 in HeLa cells depleted of NDP52 or ATG5 with siRNA ( black and grey bars are independent siRNAs ) . Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let - 7 binding sites . n = 3 independent experiments . The asterisks denote mutations in the let - 7 target sites of the reporter .

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

(b) Renilla luciferase expression controlled by let-7 in HeLa cells depleted of NDP52 or ATG5 with siRNA (black and grey bars are independent siRNAs). Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let-7 binding sites. n = 3 independent experiments. The asterisks denote mutations in the let-7 target sites of the reporter.


( c ) Western blot analysis of endogenous miRNA - repressed proteins ( RAS , HMGA2 and CDK6 are repressed by let - 7 ; CDK6 is also repressed by miR - 16 ( bottom panel ) ) or control ( TUBA , α - tubulin ) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were treated in parallel with nonspecific antagomir or let - 7 targeting antagomir .

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

(c) Western blot analysis of endogenous miRNA-repressed proteins (RAS, HMGA2 and CDK6 are repressed by let-7; CDK6 is also repressed by miR-16 (bottom panel)) or control (TUBA, α-tubulin) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were treated in parallel with nonspecific antagomir or let-7 targeting antagomir.


( d ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with control antagomir or anti - let - 7 antagomir .

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

(d) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with control antagomir or anti-let-7 antagomir.


( e ) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7 . Cells were also treated with nonspecific antagomir or let - 7 targeting antagomir .

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(e) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were also treated with nonspecific antagomir or let-7 targeting antagomir.


( f ) Renilla luciferase expression controlled by the DICER 3 ′ UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA . All error bars are s . e . m . Uncropped images of blots are shown in Supplementary Fig . S3 .

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

(f) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA. All error bars are s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.


A The first 400 nt of all 3 ' UTRs represented in the UPF1 RIP - Seq dataset were analyzed for the occurrence of motifs corresponding to the indicated RNA - binding proteins . Transcripts were divided into groups based on the number of motifs present ( 0 , 1 , more than 1 ) , indicated by the gradient triangle . The dotted line indicates the mean RIP - Seq efficiency for all transcripts used in the analysis . Mean and SD are plotted for all groups .

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

A The first 400 nt of all 3'UTRs represented in the UPF1 RIP-Seq dataset were analyzed for the occurrence of motifs corresponding to the indicated RNA-binding proteins. Transcripts were divided into groups based on the number of motifs present (0, 1, more than 1), indicated by the gradient triangle. The dotted line indicates the mean RIP-Seq efficiency for all transcripts used in the analysis. Mean and SD are plotted for all groups.


B CDF plot of relative abundance of transcripts with 3 ' UTRs > 1000 nt in K562 cells under conditions of UPF1 or non - targeting shRNA , for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations . For mRNAs with peaks > 400 nt from the TC or with peaks in the 5 ' UTR or CDS , transcripts with peaks at 3 ' UTR positions 1 - 400 were excluded . Statistical significance was determined by Kruskal - Wallis test with Dunn ' s correction for multiple comparisons and was determined based on comparison to the set with no 3 ' UTR CLIP peaks .

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

B CDF plot of relative abundance of transcripts with 3'UTRs > 1000 nt in K562 cells under conditions of UPF1 or non-targeting shRNA, for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations. For mRNAs with peaks > 400 nt from the TC or with peaks in the 5'UTR or CDS, transcripts with peaks at 3'UTR positions 1-400 were excluded. Statistical significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons and was determined based on comparison to the set with no 3'UTR CLIP peaks.


C Representative northern blots assaying the stability of β - globin reporter transcripts containing the SMG5 3 ' UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet - off cells . Half - lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint . Significance was measured compared to the SMG5 3 ' UTR reporter using the sum - of - squares F test .

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

C Representative northern blots assaying the stability of β-globin reporter transcripts containing the SMG5 3'UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet-off cells. Half-lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint. Significance was measured compared to the SMG5 3'UTR reporter using the sum-of-squares F test.


D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from ( C ) . " AS " indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3 ' UTR in the same position as the RSE . The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation . * indicates P = 0 . 0146 in two - tailed Student ' s t - test . The reporter containing the GAP 3 ' UTR used here as a transfection control contains a predicted hnRNP L binding site , allowing its consistent recovery in the IPs .

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

D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from (C). "AS" indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3'UTR in the same position as the RSE. The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation. * indicates P = 0.0146 in two-tailed Student's t-test. The reporter containing the GAP 3'UTR used here as a transfection control contains a predicted hnRNP L binding site, allowing its consistent recovery in the IPs.


A RNA - seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1 - dependent manner . B CDF plot of transcript 3 ' UTR lengths for subsets identified in ( A ) . Statistical significance was determined by two - sided K - S test . P value for decreased set is from comparison to the set of total transcripts . P value for rescued set is from comparison to decreased set .

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

A RNA-seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1-dependent manner. B CDF plot of transcript 3'UTR lengths for subsets identified in (A). Statistical significance was determined by two-sided K-S test. P value for decreased set is from comparison to the set of total transcripts. P value for rescued set is from comparison to decreased set.


A qPCR of selected transcripts found by RNA - Seq to decrease in abundance in the absence of hnRNP L . Values represent the mean and standard deviation of 3 independent biological replicates . Significance of hnRNP L KD and UPF1 KD were compared to NT , and the double knockdown was compared to hnRNP L KD . For clarity , only a single star is used to indicate significance ; in all cases P ≤ 0 . 025 in two - tailed Student ' s t - tests .

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

A qPCR of selected transcripts found by RNA-Seq to decrease in abundance in the absence of hnRNP L. Values represent the mean and standard deviation of 3 independent biological replicates. Significance of hnRNP L KD and UPF1 KD were compared to NT, and the double knockdown was compared to hnRNP L KD. For clarity, only a single star is used to indicate significance; in all cases P ≤ 0.025 in two-tailed Student's t-tests.


B Half - lives of selected transcripts under conditions of non - targeting or hnRNP L knockdown . Measurements were calculated based on proportional recovery of transcript labeled with 5 - EU in 1 hour versus total transcript . Significance was determined using two - tailed Student ' s t - test : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 . Values represent mean and standard deviation of 3 independent replicates .

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B Half-lives of selected transcripts under conditions of non-targeting or hnRNP L knockdown. Measurements were calculated based on proportional recovery of transcript labeled with 5-EU in 1 hour versus total transcript. Significance was determined using two-tailed Student's t-test: *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Values represent mean and standard deviation of 3 independent replicates.


C The stability of β globin reporters with the CSRP1 3 ' UTR , along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated , were evaluated in HeLa Tet - off cells . The schematic indicates areas of 3 ' UTR CA - enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3 ' UTR , where putative hnRNP L binding elements are in red typeface . The northern blot is a representative example from 3 independent replicates . Mean values plotted with SD at each timepoint were used to quantify decay ( right panel ) . Significance is measured compared to the WT reporter using the sum - of - squares F test .

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

C The stability of β globin reporters with the CSRP1 3'UTR, along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated, were evaluated in HeLa Tet-off cells. The schematic indicates areas of 3' UTR CA-enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3'UTR, where putative hnRNP L binding elements are in red typeface. The northern blot is a representative example from 3 independent replicates. Mean values plotted with SD at each timepoint were used to quantify decay (right panel). Significance is measured compared to the WT reporter using the sum-of-squares F test.


D The stability of the WT and L mut 1 - 200 CSRP1 reporters in HeLa Tet - off cells treated with non - targeting RNAi or siRNA against UPF1 was evaluated . The northern blot is a representative example from at least 3 biological replicates . Decay quantification and statistics were performed as in ( C ) , with significance measured compared to the non - targeting condition .

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

D The stability of the WT and L mut 1-200 CSRP1 reporters in HeLa Tet-off cells treated with non-targeting RNAi or siRNA against UPF1 was evaluated. The northern blot is a representative example from at least 3 biological replicates. Decay quantification and statistics were performed as in (C), with significance measured compared to the non-targeting condition.


A Motif analysis of recovered UPF1 - associated transcripts analyzed by RIP - Seq Transcripts were binned into three 3 ' UTR length classes , which were then further subdivided based on hnRNP L motif density . Each box plot represents the median and interquartile range with extensions from 10 % to 90 % of the indicated subset . Dotted line represents the median RIP - Seq efficiency of the class of transcripts with the shortest 3 ' UTR and no hnRNP L binding motifs . Significance was computed using Kruskal - Wallis tests with Dunn ' s correction for multiple comparisons . B CDF plots of selected transcript classes from ( A ) . Log2 ( FC ) of UPF1 knockdown as compared to the non - targeting control indicates NMD sensitivity of each class . Significance was determined by Kruskal - Wallis test with Dunn ' s correction for multiple comparisons Data information : * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq Transcripts were binned into three 3'UTR length classes, which were then further subdivided based on hnRNP L motif density. Each box plot represents the median and interquartile range with extensions from 10% to 90% of the indicated subset. Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and no hnRNP L binding motifs. Significance was computed using Kruskal-Wallis tests with Dunn's correction for multiple comparisons. B CDF plots of selected transcript classes from (A). Log2(FC) of UPF1 knockdown as compared to the non-targeting control indicates NMD sensitivity of each class. Significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons Data information: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C Motif analysis of recovered UPF1 - associated transcripts analyzed by RIP - Seq Transcripts are divided into tertiles by hnRNP L and PTBP1 binding motif density and 3 ' UTR length . Data are represented and analyzed as in ( A ) . Dotted line represents the median RIP - Seq efficiency of the class of transcripts with the shortest 3 ' UTR and low PTBP1 / hnRNP L motif density . D CDF plots of selected transcript classes from ( C ) analyzed as in ( B ) . Data information : * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq Transcripts are divided into tertiles by hnRNP L and PTBP1 binding motif density and 3'UTR length. Data are represented and analyzed as in (A). Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and low PTBP1/hnRNP L motif density. D CDF plots of selected transcript classes from (C) analyzed as in (B). Data information: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C qPCR of selected transcripts in SU - DHL - 4 cells under conditions of non - targeting RNAi or hnRNP L knockdown with or without 50 μg / mL cycloheximide treatment ( for 4 hours ; n ≥ 3 ) . Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO , and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO . Bars represent the mean ± SD . For clarity , only a single star was used to indicate significance ; in all cases P ≤ 0 . 05 in two - tailed Student ' s t - tests .

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

C qPCR of selected transcripts in SU-DHL-4 cells under conditions of non-targeting RNAi or hnRNP L knockdown with or without 50 μg/mL cycloheximide treatment (for 4 hours; n ≥ 3). Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO, and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO. Bars represent the mean ± SD. For clarity, only a single star was used to indicate significance; in all cases P ≤ 0.05 in two-tailed Student's t-tests.


E Half - life determination from RT - qPCR detection of reporter constructs in HeLa Tet - off cells transfected with non - targeting or UPF1 siRNAs . For plots comparing non - targeting with UPF1 knockdown , significance is compared to the non - targeting condition using the the sum - of - squares F test . The fourth plot compares the non - targeting decay curves of the three reporters , and significance is compared to the decay of the fusion transcript . Points represent the mean ± SD from qRT - PCR at each time point of at least 3 independent experiments .

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

E Half-life determination from RT-qPCR detection of reporter constructs in HeLa Tet-off cells transfected with non-targeting or UPF1 siRNAs. For plots comparing non-targeting with UPF1 knockdown, significance is compared to the non-targeting condition using the the sum-of-squares F test. The fourth plot compares the non-targeting decay curves of the three reporters, and significance is compared to the decay of the fusion transcript. Points represent the mean ± SD from qRT-PCR at each time point of at least 3 independent experiments.


A qPCR of selected transcripts in SU - DHL - 4 cells under conditions of BCL2 knockdown , hnRNP L knockdown , or non - targeting control siRNA Nucleofection ( n ≥ 4 independent experiments ) . Bars indicate mean ± SD . Significance was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A qPCR of selected transcripts in SU-DHL-4 cells under conditions of BCL2 knockdown, hnRNP L knockdown, or non-targeting control siRNA Nucleofection (n ≥ 4 independent experiments). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


Quantification of protein reduction in knockdown experiments and a representative western blot . Each point represents a trial ( n ≥ 5 for a given condition ) . Mean with standard deviation are indicated on the plot . Target protein levels from all biological replicates were normalized to MATRIN3 , with statistical analysis as in panel ( A ) . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

Quantification of protein reduction in knockdown experiments and a representative western blot. Each point represents a trial (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot. Target protein levels from all biological replicates were normalized to MATRIN3, with statistical analysis as in panel (A). Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


C FACS - based apoptosis analysis of SU - DHL - 4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours . Quantification of relative change in each quadrant is provided where points represent individual experiments ( n ≥ 5 for a given condition ) . Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel ( A ) . A representative FACS experiment is shown with the percentage of cells in each gate indicated . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O B-EXP_ASSAY O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS-based apoptosis analysis of SU-DHL-4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours. Quantification of relative change in each quadrant is provided where points represent individual experiments (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel (A). A representative FACS experiment is shown with the percentage of cells in each gate indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.


B SU - DHL - 4 and SU - DHL - 6 cells were treated with varying doses of BCL2 inhibitor . Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells . Traces were generated from at least 5 independent biological replicates . Points represent mean ± SD at each dose . P value from comparison of dose response between SU - DHL - 4 and SU - DHL - 6 cells calculated using sum - of - squares F test is given in the graph key . Significance of individual points calculated using two - tailed Student ' s t - tests is marked using asterisks on the graph . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

B SU-DHL-4 and SU-DHL-6 cells were treated with varying doses of BCL2 inhibitor. Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells. Traces were generated from at least 5 independent biological replicates. Points represent mean ± SD at each dose. P value from comparison of dose response between SU-DHL-4 and SU-DHL-6 cells calculated using sum-of-squares F test is given in the graph key. Significance of individual points calculated using two-tailed Student's t-tests is marked using asterisks on the graph. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


C qPCR of selected transcripts in SU - DHL - 6 cells under conditions of hnRNP L knockdown or non - targeting control ( n = 5 independent experiments ) . Bars indicate mean ± SD . Significance of hnRNP L KD was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

C qPCR of selected transcripts in SU-DHL-6 cells under conditions of hnRNP L knockdown or non-targeting control (n = 5 independent experiments). Bars indicate mean ± SD. Significance of hnRNP L KD was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


D Representative amplification of the region of chromosomal deletion in edited cells . PCR products were run on a 1 % agarose gel to resolve the amplicons from edited and unedited chromosomes . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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D Representative amplification of the region of chromosomal deletion in edited cells. PCR products were run on a 1% agarose gel to resolve the amplicons from edited and unedited chromosomes. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


E qPCR of selected transcripts in edited SU - DHL - 6 cells ( n = 4 independent experiments ) . Significance was determined as compared to control Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

E qPCR of selected transcripts in edited SU-DHL-6 cells (n = 4 independent experiments). Significance was determined as compared to control Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells . Target protein levels from all biological replicates were normalized to MATRIN3 . Each point represents a trial ( n = 4 ) . Bars indicate mean ± SD . Significance was determined compared to NT using two - tailed Student ' s t - tests . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells. Target protein levels from all biological replicates were normalized to MATRIN3. Each point represents a trial (n = 4). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


G FACS - based apoptosis analysis of edited SU - DHL - 6 . Quantification of relative change in each quadrant is provided , where points represent individual experiments ( n = 6 for a given condition ) . Mean with standard deviation are indicated on the plot , analyzed as in ( F ) . Shown is a representative FACS experiment . Percentage of cells in each gate is indicated . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

G FACS-based apoptosis analysis of edited SU-DHL-6. Quantification of relative change in each quadrant is provided, where points represent individual experiments (n = 6 for a given condition). Mean with standard deviation are indicated on the plot, analyzed as in (F). Shown is a representative FACS experiment. Percentage of cells in each gate is indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8 were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) ,

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A),


( A , B ) Wild - type ( WT ) ( BY4741 ) cells expressing GFP − Atg8were grown to mid - log - phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or with DMSO ( - ) in the rich medium for 30 min . Cells were washed and shifted to nitrogen - starvation medium ( SD - N ) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0 . 1 mM palmitic / stearic / myristic acids or DMSO . Cells were then lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies ( A ) , or were visualized by fluorescence microscopy ( B )

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

(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)


( C ) Cells ( TOS038 ) expressing Atg1 - GFP were grown to mid - log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min . Cells were washed , shifted to SD - N for 4 h , and then visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( BY4741 ) and pep4 ∆ ( TOS015 ) strains were grown to mid - log phase and preincubated in YPD with cerulenin or DMSO for 30 min . Cells were pulse - labeled for 10 min with [ 35S ] methionine and cysteine and chased for the indicated time periods . Cell lysates were subjected to immunoprecipitation with anti - Ape1 antibodies followed by SDS ‒ PAGE and X - ray film to detect radioactive signals . prApe1 , premature Ape1 ; mApe1 , mature Ape1 . (

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

(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (


( E ) Cells of the fas1 ∆ ( TOS029 ) strain expressing GFP ‒ Atg8 were grown to mid - log phase in YPD + 0 . 1 mM palmitic / stearic / myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min . Cells were then shifted to SD - N for the indicated times . Cell lysates were subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies .

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

(E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.


( F ) WT ( BY4741 ) cells were grown to mid - log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin ( 25 , 50 ) or with DMSO ( - ) in rich medium for 30 min . Cells were washed and shifted to SD - N in the presence of cerulenin ( 25 , 50 ) or DMSO ( - ) . Cells were lysed and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP and anti - Pgk1 antibodies . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type YPD , complete medium .

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

(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.


( A ) Cells ( TN124 strain ) were grown to mid - log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods ( Cer preincubation ) or incubated with DMSO without preincubation ] ( - ) . Cells were then shifted to SD - N for 3 h with the addition of DMSO or 50 µM cerulenin ( cer ) . Autophagic activity was measured by alkaline phosphatase assay . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) Cells were grown as in ( A ) , stained with BODIPY , and visualized by fluorescence microscopy . Scale bar , 5 μm .

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(B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm.


( C ) WT ( SCY62 ) and tagΔsteΔ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 3 h . Cells were stained with Nile red and visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.


( D ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cell lysates were subjected to SDS − PAGE , followed by western blot analysis using anti - GFP , anti - Ape1 ( prApe1 , premature Ape1 ; mApe1 , mature Ape1 ) , and anti - Fas1 and anti - Pgk1 antibodies .

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

(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.


( E ) WT ( SCY62 ) and tag ∆ ste ∆ ( H1246 ) cells were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . cer , cerulenin ; DIC , Differential interference contrast ; FA , fatty acids ; SD - N , nitrogen - starvation medium ; WT , wild - type , YPD , complete medium .

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

(E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.


( A ) GAL - DGA1GAL - ARE2lro1Δ are1Δ ( FYS118 strain ) was grown on SC ( synthetic minimal medium without dextrose ) + raffinoseovernight , diluted to OD 0 . 4 , and grown to mid - log phase in either SC + glucose or SC + galactose medium . Cells were shifted to SD - N for 2 h , stained with BODIPY , and visualized by fluorescencemicroscopy . Scale bar , 5 μm .

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

(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.


( B ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 were grown as in A . Cells were lysed at the indicated times and subjected to SDS ‒ PAGE , followed by western blot analysis using anti - GFP antibodies . (

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

(B) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (


( C , D ) WT ( BY4741 ) and GAL - DGA1 GAL - ARE2 lro1 ∆ are1 ∆ ( FYS118 ) cells expressing GFP − Atg8 ( C ) or GFP − Atg1 ( D ) were grown as in A and visualized by fluorescence microscopy after 2 h in SD - N . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild - type .

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

(C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 6 h . Cells were stained with BODIPY and visualized by fluorescence microscopy . Scale bar , 5 μm .

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

(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.


( A ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . ( B ) Quantification of the GFP / GFP − Atg8 ratio . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

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

(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


. ( C ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells expressing GFP - Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 2 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . (

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

. (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. (


( D ) WT ( SCY62 ) , tag ∆ ste ∆ ( H1246 ) , tag ∆ ( H1226 ) and ste ∆ ( H1112 ) cells were grown as in ( A ) . Lysates were subjected to SDS − PAGE in urea gel , followed by western blot analysis using anti - Atg8 and anti - Pgk1 antibodies . Atg8 I , non - lipidated Atg8 ; Atg8 II , lipidated Atg8 . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


Fig . 6 Lack of lipid droplets inhibits starvation - induced formation of autophagosomes . ( A , H ) WT pep4 ∆ ( pep4 ∆ ) ( A , B ) , tag ∆ ste ∆ pep4 ∆ ( tag ∆ ste ∆ pep4 ∆ ) ( C , D ) , ste ∆ pep4 ∆ ( ste ∆ pep4 ∆ ) ( E , F ) and tag ∆ pep4 ∆ ( tag ∆ pep4 ∆ ) ( G , H ) cells were grown to an exponential phase in YPD before being starved in SD - N for 2 h , and were then processed for electron microscopy . H is a magnification of the box in panel G . Magnification of proliferating ER in a tag ∆ ste ∆ ( H1246 ) cell is shown in ( I ) Asterisks indicate autophagic bodies . N , nucleus ; PM , plasma membrane ; CW , cell wall ; ER , endoplasmic reticulum ; M , mitochondrion ; V , vacuole ; # , trehalose ; F , ER proliferation . Scale bars : 1 µm ( A - H ) and 200 nm ( I ) . ( J ) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles . Error bars represent standard deviations fromcounting of the three grids . * * * P < 0 . 001 ( Student ' s t - test ) . SD - N , nitrogen - starvation medium ; WT , wild type YPD , complete medium .

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

Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.


( A ) WT ( BY4741 ) , tgl1 ∆ , yeh1 ∆ , and yeh2 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for the indicated time periods . Cells were lysed and subjected to SDS − PAGE , followed by western blot analysis using anti - GFP antibodies . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

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

(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( B ) WT ( BY4741 ) and tgl3 ∆ tgl4 ∆ tgl5 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) . (

O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (


( C ) WT ( BY4741 ) , ayr1 ∆ and ldh1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 ( Student ' s t - test ) .

O O O O O B-ORGANISM O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).


( D ) WT ( BY4741 ) , ldh1 ∆ ayr1 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * * P < 0 . 01 ( Student ' s - t test ) .

O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student's-t test).


( E ) WT ( BY4741 ) , ice2 ∆ and ldb16 ∆ cells expressing GFP − Atg8 were grown , treated , lysed , and western blotted as in ( A ) . Quantification of the GFP / GFP − Atg8 ratio is presented on the right . Error bars represent the s . e . m . of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ( Student ' s t - test ) .

O O O O O B-ORGANISM O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student's t-test).


( F ) WT ( BY4741 ) , ice2 ∆ and yeh1 ∆ cells expressing GFP − Atg8 were grown to mid - log phase in YPD and shifted to SD - N for 4 h . GFP − Atg8 was visualized by fluorescence microscopy . Scale bar , 5 μm . DIC , Differential interference contrast ; SD - N , nitrogen - starvation medium ; WT , wild type .

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

(F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.


( A ) Total lysates of different mouse organs underwent immunoblotting analysis using anti - Pdia4 and anti - actin antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


( C ) Min6 cells were treated with glucose and palmitate at the indicated dosages . Total lysate underwent immunoblotting analysis using the indicated antibodies . ( D ) Human islets were treated with glucose and palmitate at the indicated dosages , followed by immunoblotting analysis . ( Data information : Data from 3 experiments are presented as the mean ± SD .

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

(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.


( G ) Sera from wild - type ( WT ) BKS and Leprdb / db mice , aged 4 , 8 , and 18 weeks , B6 mice , fed with a normal diet ( ND ) and a high fat diet ( HFD ) for 4 weeks , and humans , healthy volunteers and diabetic patients ( T2D ) , were quantified for Pdia4 level using an anti - Pdia4 ELISA kit . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-TISSUE O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-DISEASE O O B-DISEASE O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( H ) Immunoblotting analysis of Pdia4 and markers in the cytosolic ( Cyto ) , nuclear ( Nuc ) , membrane ( Mem ) , mitochondrial ( Mito ) , and ER compartments of Ming 6 cells using the indicated antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


( A ) Survival rate and life span of WT , Pdia4 - / - ( KO ) , and Pdia4tg / tg ( TG ) mice on Leprdb / db background from birth to 120 weeks Data information : The number of mice ( n ) is indicated in parentheses . Data from over 3 experiments are presented as the mean ± SD . Log rank test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival rate and life span of WT, Pdia4-/-(KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks Data information: The number of mice (n) is indicated in parentheses. Data from over 3 experiments are presented as the mean ± SD. Log rank test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of 18 - week - old WT and Pdia4 - / - ( KO ) mice on BKS or Leprdb / db background were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pancreata of 18-week-old WT and Pdia4-/- (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The same batch mice as in ( B ) were given water containing BrdU . The BrdU + cells of the islets were visualized and quantified ( BrdU + labeling ) . TUNEL - positive cells in the islets of the mice ( B ) were visualized and quantified ( TUNEL assay ) . BrdU + cells per islet area ( 0 . 01 mm2 ) and TUNEL + cells per islet area ( 0 . 05 mm2 ) are expressed in arbitrary units ( AU ) . The dash circles indicate islet regions and the black arrowheads indicate TUNEL + cells . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions and the black arrowheads indicate TUNEL+ cells. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


F ) insulin in supernatants of the mouse islets ( F ) in GSIS assays . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 ) were used in GSIS assays . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) insulin in supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) were used in GSIS assays. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of the same batch of mice as in ( A ) , at the age of 14 weeks , were stained with anti - insulin ( αIns ) antibody and DHE ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar = 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The islets of the mice from ( C ) were tested for GSIS . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 mM ) were used in GSIS assays . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) The islets of WT , Pdia4 - / - ( KO ) and Pdia4tg / tg ( TG ) BKS mice were isolated and grown in complete DMEM medium containing 3 . 3 mM ( LG ) and 30 mM glucose ( HG ) for 12 h . The islets stained with propidium iodide ( PI ) , photographed , and quantified . Scale bar = 50 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(A) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The islets stained with propidium iodide (PI), photographed, and quantified. Scale bar = 50 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . Mitochondrial ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O B-CELL_TYPE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The islets from the mice (A) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with Hoechst 33342 ( Ho ) plus CM - H2DCFDA in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . cytosolic ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O B-CELL_TYPE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The islets from the mice (A) were incubated with Hoechst 33342 (Ho) plus CM-H2DCFDA in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). cytosolic ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( E ) Mitochondria isolated from the mouse islets ( A ) were measured for the activity of ETC CI ( left ) , CII / III ( middle ) and CIV ( right ) using MitoCheck assays . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(E) Mitochondria isolated from the mouse islets (A) were measured for the activity of ETC CI (left), CII/III (middle) and CIV (right) using MitoCheck assays. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) Ultrastructure of mitochondria and insulin granules in typical β - cells of 3 mouse lines on BKS and Leprdb / db backgrounds . Arrows indicate the mitochondria whereas arrowheads indicate insulin granules . Scale bar = 1 μm . Data information : A total of 15 - 23 images per group were analyzed . Data from each group are presented as the mean ± SD .

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

(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD.


( E ) The construct encoding Flag - Pdia4 or its mutants and that expressing Myc / Flag - tagged Ndufs3 ( left ) or p22phox ( right ) were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis . N - terminal Flag tag , catalytic domains ( a , a ' and a ' ' ) , non - catalytic domains ( b and b ' ) , and C - terminal KEEL are indicated . Pdia4 * is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs ( a * , a ' * and a ' ' * ) .

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

(E) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).


( F ) The construct encoding Myc / Flag - tagged p22phox , Ndufs3 or their mutants and that expressing Flag - tagged Pdia4 were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis using anti - Flag antibody .

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

(F) The construct encoding Myc/Flag-tagged p22phox, Ndufs3 or their mutants and that expressing Flag-tagged Pdia4 were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis using anti-Flag antibody.


( A ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) , a Pdia4 RNAi ( KD ) and a Pdia4 cDNA ( OVE ) were sorted and tested for the Pdia4 protein level ( left ) . The cells were incubated with NAC ( 1 mM ) and then stained with MitoSOX or CellROX in response to 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose for an additional 30 min . Signal from MitoSOX ( middle ) and CellROX ( right ) was re - plotted into histograms . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) and an RNAi of Ndufs3 ( Ndufs3 KD ) or p22phox ( p22phox KD ) were selected and tested for levels of Ndufs3 ( 1st column ) , mitochondrial ROS ( 2nd column , MitoSOX ) , p22phox ( 3rd column ) , and cytosolic ROS ( 4th column , CellROX ) . The cells were grown in the presence of 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose . ( C ) The same experiments as ( B ) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 ( tNdufs3 ) and p22phox ( tp22phox ) . ( Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) 293T cells , which were transfected with the construct encoding Flag - Pdia4 and that expressing Myc / Flag - tagged p22phox or Ndufs3 , were treated with GHTT ( 28 μM ) for 30 min . The cells were lysed and incubated with anti - Pdia4 antibodies plus protein G beads . Their total lysates ( TL ) and immunoprecipitates ( IP ) underwent immunoblotting analysis with anti - Flag antibody .

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

(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody.


( F ) The sections of pancreata of the mice ( E ) were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O B-TISSUE O O B-ORGANISM O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


C , Microscale thermophoresis ( MST ) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711 . D , dDATmfc did not show any interactions with NO711 in the MST experiments . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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

C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions with NO711 in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


E , SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM . F , MST experiments display no interactions of purified dDATmfc with SKF89976a . The MST profiles shown are one of two independent measurements . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


G , Michaelis - Menten uptake kinetics of 3H - GABA by the GAT1WT used in the study displaying a KM value of 11 . 4 μM .

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

G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.


H , Inhibition of 3H - GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14 . 4 μM . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


I , Inhibition of 3H - GABA uptake through GAT1WT by tiagabine ( Ki = 725 nM ) , NO711 ( 1 . 07 μM ) and SKF89976a ( 7 . 3 μM ) . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM). Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.