Figure 5 ( D ) qPCR revealed the fold change ( UPF1 knockdown vs control ) of candidate genes in ( C ) with G1 down - expression depending on NMD ( n = 3 ) , the region above the dashed line means that depleting UPF1 increases the mRNA level of the candidate genes . Data are presented as mean ± s . d . in ( D ) two - sided unpaired t - test is used to determine the differences . ( E ) Analysis of different AS events in 4 candidate gene showed in ( D ) by sqRT - PCR ( n = 3 ) , bar plot showing the ratio of WT isoform , the above is the representative DNA gel . Data are presented as mean ± s . d . in ( E ) , a two - sided unpaired t - test is used to determine the differences . ( J to M ) S vs G1 phase expression difference ( J , M ) and gene expression levels in S phase ( K , L ) of AS - NMD regulated S vs G1 phase up - regulated genes and other S vs G1 phase up - regulated genes ( control ) by RNA - Seq analysis , showed as log2 fold change in ( J ) , TPM in ( K ) and ( M ) , or cumulative distribution function charts in ( L ) . TPM , transcripts per kilobase million . Ctrl , control For the RNA - seq data used n = 3 biological replicate . For ( J ) , ( K ) and ( M ) , the central band represent the median , the boxes represent the first and third quartiles , and the whiskers is based on the 1 . 5 IQR ( Interquartile range ) . For ( J - M ) , Mann - Whitney U test is used to determine the differences .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(D) qPCR revealed the fold change (UPF1 knockdown vs control) of candidate genes in (C) with G1 down-expression depending on NMD (n = 3), the region above the dashed line means that depleting UPF1 increases the mRNA level of the candidate genes . Data are presented as mean ± s.d. in (D) two-sided unpaired t-test is used to determine the differences.(E) Analysis of different AS events in 4 candidate gene showed in (D) by sqRT-PCR (n = 3), bar plot showing the ratio of WT isoform, the above is the representative DNA gel. Data are presented as mean ± s.d. in (E), a two-sided unpaired t-test is used to determine the differences.(J to M) S vs G1 phase expression difference (J, M) and gene expression levels in S phase (K, L) of AS-NMD regulated S vs G1 phase up-regulated genes and other S vs G1 phase up-regulated genes (control) by RNA-Seq analysis, showed as log2 fold change in (J), TPM in (K) and (M), or cumulative distribution function charts in (L). TPM, transcripts per kilobase million. Ctrl, control For the RNA-seq data used n = 3 biological replicate. For (J), (K) and (M), the central band represent the median, the boxes represent the first and third quartiles, and the whiskers is based on the 1.5 IQR (Interquartile range). For (J-M), Mann-Whitney U test is used to determine the differences.


Figure 6γH2AX levels in RBM10 - KD # 4 cells under the treatments as indicated . Representative images were shown in ( A ) . CPT , camptothecin . BLM , bleomycin . IR , ionizing radiation . Immunofluorescence analysis of γH2AX in RBM10 - KD # 4 cells complemented with either RBM10 . ( H and I ) HeLa cells were transfected with RBM10 or / with 53BP1 siRNA . Immunofluorescence analysis of γH2AX after CPT ( H ) and cisplatin ( I ) treatment . Colony formation assay ( n = 3 ) was performed in RBM10 - depleted 293T cells treated with ionizing radiation ( n = 3 ) ( K ) ( M ) Immunofluorescence analysis of γH2AX in 293T cells expressing the indicated variants in G1 phase .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 6γH2AX levels in RBM10-KD #4 cells under the treatments as indicated. Representative images were shown in (A). CPT, camptothecin. BLM, bleomycin. IR, ionizing radiation.Immunofluorescence analysis of γH2AX in RBM10-KD #4 cells complemented with either RBM10.(H and I) HeLa cells were transfected with RBM10 or/with 53BP1 siRNA. Immunofluorescence analysis of γH2AX after CPT (H) and cisplatin (I) treatment.Colony formation assay (n = 3) was performed in RBM10-depleted 293T cells treated with ionizing radiation (n = 3) (K)(M) Immunofluorescence analysis of γH2AX in 293T cells expressing the indicated variants in G1 phase.


Figure 7Immunohistochemical assay was performed to determinate RBM10 and BRCA1 levels in ovarian cancer microarray samples . Representative images are shown in ( B ) . Colony formation assay ( n = 3 ) for the determination of the viability of SK - OV - 3 ( F ) , HeLa ( G ) cells treated with a combination of SB1317 and Olaparib . SB1317 , CDK2 inhibitor . Statistical analysis results of tumor weight and sizes 21 days post seeding are shown in ( J ) and ( K ) .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 7Immunohistochemical assay was performed to determinate RBM10 and BRCA1 levels in ovarian cancer microarray samples. Representative images are shown in (B).Colony formation assay (n = 3) for the determination of the viability of SK-OV-3 (F), HeLa (G) cells treated with a combination of SB1317 and Olaparib. SB1317, CDK2 inhibitor.Statistical analysis results of tumor weight and sizes 21 days post seeding are shown in (J) and (K).


Figure 1 ( C , D ) Standard replication assays on undamaged ( UD ) , thymine glycol ( Tg ) , and cyclobutane pyrimidine dimer ( CPD ) templates in the presence ( C ) and absence ( D ) of Pol η , Pol ζ , Rev1 , Rad6 - Rad18 , Uba1 , and ubiquitin ( TLS machinery ) . Reactions contained 2 . 5 nM Pol δ .

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

Figure 1 (C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ.


Figure 2 ( B ) Oligonucleotide competition assay on UD and Tg templates . S ; scrambled oligonucleotide . ( C ) Lane profiles of lanes 4 - 6 in ( B ) normalised to the run off product . FL ; FL - lead , OR ; oligonucleotide restart , S ; Stall , RO ; Run off , OF ; Okazaki fragments . ( D ) Pulse chase reaction on UD and Tg templates performed as shown . Where present , Pol δcat ( 10 nM ) was added directly after addition of the chase . ( E ) Standard replication reaction performed in the absence and presence of RFC / PCNA on the Tg template .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 2 (B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide. (C) Lane profiles of lanes 4-6 in (B) normalised to the run off product. FL; FL-lead, OR; oligonucleotide restart, S; Stall, RO; Run off, OF; Okazaki fragments.  (D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase. (E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.


Figure 3 ( B ) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel . ( D ) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel . ( E ) Titration of Pol δ in a pulse chase reaction on the Tg template . Pol δ was added after the 0 min time point was taken . ( F ) Quantification of bypass 5 min after addition of the chase for the data in ( E ) . ( G ) Titration of Pol δ or Pol ε ( 1 , 2 . 5 , 5 , 7 . 5 , 10 nM ) into a primer extension assay on a Tg template in the presence of PCNA and RFC . The primer / template sequence and location of the lesion are shown above .

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

Figure 3 (B) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.  (D) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel.  (E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken. (F) Quantification of bypass 5 min after addition of the chase for the data in (E).  (G) Titration of Pol δ or Pol ε (1, 2.5, 5, 7.5, 10 nM) into a primer extension assay on a Tg template in the presence of PCNA and RFC. The primer/template sequence and location of the lesion are shown above.


Figure 4 ( B ) Mapping the 3ʹ - end of the stalled nascent leading strand . Pulse chase reaction on Tg and CPD templates . Where present , Pol δ ( 10 nM ) was added immediately after the 0 min time point . Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand . dd ; dideoxy .

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

Figure 4 (B) Mapping the 3ʹ-end of the stalled nascent leading strand. Pulse chase reaction on Tg and CPD templates. Where present, Pol δ (10 nM) was added immediately after the 0 min time point. Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand. dd; dideoxy.


Figure 5 ( A ) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP , dCTP , dGTP , or dTTP . ( C ) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in ( B ) . Three individual experiments were performed , two in the presence and one in the absence of Pol δ ( 10 nM ) , giving 99 , 97 , and 95 successful sequencing reads respectively .

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

Figure 5 (A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP.  (C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively.


Figure 6 ( A ) Standard replication assay on undamaged ( UD ) , 8 - oxoguanine ( 8oxoG ) , and thymine glycol ( Tg ) templates in the absence of TLS components . ( B ) Pulse chase reaction on UD and 8oxoG templates . Where present , Pol δcat ( 10 nM ) was added directly after addition of the chase . ( C ) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Standard replication assay on undamaged (UD), 8-oxoguanine (8oxoG), and thymine glycol (Tg) templates in the absence of TLS components. (B) Pulse chase reaction on UD and 8oxoG templates. Where present, Pol δcat (10 nM) was added directly after addition of the chase.  (C) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.


Figure 1A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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.


Figure 2A 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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.


Figure 3A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A 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.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.


Figure 4A 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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.


Figure 5C 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 .

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Figure 5C 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.


Figure 6A 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 . 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 .

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Figure 6A 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.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.


Figure 7B 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 . 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 . 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B 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.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.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.


Figure 1E . Ribosomal pull down shows lack of occupancy of ribosomes on veal2 . fli1a and actb were used as positive controls . F - I . e - GFP fusion assay confirms lack of peptide formation from veal2 sequence . ( F - G ) mitfa - eGFP fused transcript . ( H - I ) veal2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected embryos . Scale bar - 100μm .

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Figure 1E. Ribosomal pull down shows lack of occupancy of ribosomes on veal2. fli1a and actb were used as positive controls.F-I. e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (F-G) mitfa-eGFP fused transcript. (H-I) veal2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μm.


Figure 2F - T . Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8 / + zebrafish upon complementing with the wild type ( WT ) veal2 RNA . ( F - J ) Control zebrafish embryos . ( K - O ) veal2gib005Δ8 / + embryos injected with vehicle control . ( P - T ) veal2gib005Δ8 / + embryos complemented with veal2 RNA . ( F , K , P ) bright field . ( G , L , Q ) mRFP . ( H , M , R ) Animals stained with O - dianisidine stain . ( I , N , S ) eGFP . ( J , O , T ) merged eGFP and mRFP filters . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . ( F - H , K - M , P - R ) magnification - 5X , scale bar - 100μm ( I - J , N - O , S - T ) magnification - 20X , Scale bar - 50μm .

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Figure 2F-T. Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8/+ zebrafish upon complementing with the wild type (WT) veal2 RNA. (F-J) Control zebrafish embryos. (K-O) veal2gib005Δ8/+ embryos injected with vehicle control. (P-T) veal2gib005Δ8/+ embryos complemented with veal2 RNA. (F,K,P) bright field. (G,L,Q) mRFP. (H,M,R) Animals stained with O-dianisidine stain. (I,N,S) eGFP. (J,O,T) merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F-H,K-M,P-R) magnification-5X, scale bar-100μm (I-J,N-O,S-T) magnification-20X, Scale bar-50μm.


Figure 3C . RNA immunoprecipitation of veal2 was performed by pull - down of Prkcbb and Epbhb3 , followed by qRT - PCR . IgG pull - down was performed as control . Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples , along with 10 % input control , as marked by arrowheads . D . qRT - PCR based quantification of veal2 transcript across Prkcbb , Ephb3 and IgG immunoprecipitations . actb was used as normalization control . Data from three different biological replicates represented as mean fold change values ± standard deviation . F - K . Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8 / + zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8 / + . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure . ( F - I ) Magnification - 5X , Scale bar - 100μm , ( J - K ) Magnification - 20X , Scale bars - 50μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.D. qRT-PCR based quantification of veal2 transcript across Prkcbb, Ephb3 and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation.F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm.


Figure 4F . Abundance of Prkcbb protein in total cell ( T ) , cytoplasmic ( C ) and membrane ( M ) fractions of cells from 2 dpf gib004Tg ( fli1a : EGFP ; gata1a : DsRed ) and veal2gib005Δ8 / + zebrafish embryos . Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8 / + zebrafish embryos .

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Figure 4F. Abundance of Prkcbb protein in total cell (T), cytoplasmic (C) and membrane (M) fractions of cells from 2 dpf gib004Tg(fli1a:EGFP;gata1a:DsRed) and veal2gib005Δ8/+ zebrafish embryos. Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8/+ zebrafish embryos.


Figure 5E - H e - GFP fusion assay confirms lack of peptide formation from veal2 sequence . ( E - F ) mitfa - eGFP fused transcript . ( G - H ) VEAL2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected embryos . Scale bar - 100μI - N . Complementation of VEAL2 in veal2gib005Δ8 / + embryos significantly rescued hemorrhage phenotype . Arrowheads show the presence of hemorrhage due to the vascular integrity defects . ( I - L ) Magnification - 5X , Scale bar - 100μm ( M - N ) Magnification - 20X , Scale bars - 50μm . R - T . Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization . ( R ) PRKCB in the GFP channel . ( S ) VEAL2 in CAL - fluor red ( 610nM ) . ( T ) Merged image for PRKCB and VEAL2 . Magnification - 100X , scale bar - 20μm .

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Figure 5E-H e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (E-F) mitfa-eGFP fused transcript. (G-H) VEAL2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μI-N. Complementation of VEAL2 in veal2gib005Δ8/+ embryos significantly rescued hemorrhage phenotype. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I-L) Magnification-5X, Scale bar-100μm (M-N) Magnification-20X, Scale bars-50μm.R-T. Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL-fluor red (610nM). (T) Merged image for PRKCB and VEAL2. Magnification-100X, scale bar-20μm.


Figure 6K - AB . VEAL2 regulates junctional dynamics by interacting with PRKCB . Overexpression of VEAL2 retains PRKCB mostly in cytoplasm and keeps strong junctional assembly formation of CDH5 and CTNNB1 on the membrane . Knockdown of VEAL2 led to migration of PRKCB on membrane and henceforth degradation of junctional assembly of CDH5 and CTNNB1 . ( K - N , W - X ) PRKCB . ( O - R , Y - Z ) CDH5 . ( S - V , AA - AB ) CTNNB1 . ( K - V ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of proteins in HUVECs . ( W - AB ) Dot plot representing quantification of protein signal localization in membrane / total fraction . The quantification was done using imageJ . Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as individual values ; the middle bar represents the mean and the error bar represents ± standard deviation .

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Figure 6K-AB. VEAL2 regulates junctional dynamics by interacting with PRKCB. Overexpression of VEAL2 retains PRKCB mostly in cytoplasm and keeps strong junctional assembly formation of CDH5 and CTNNB1 on the membrane. Knockdown of VEAL2 led to migration of PRKCB on membrane and henceforth degradation of junctional assembly of CDH5 and CTNNB1. (K-N,W-X) PRKCB. (O-R,Y-Z) CDH5. (S-V,AA-AB) CTNNB1. (K-V) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (W-AB) Dot plot representing quantification of protein signal localization in membrane/total fraction. The quantification was done using imageJ. Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


Figure 7E - S . Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein . Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia . ( E - H , Q ) CDH5 protein , ( I - L , R ) CTNNB1 protein , ( M - P , S ) PRKCB protein . ( E - P ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of proteins in HUVECs . ( Q - S ) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as individual values ; the middle bar represents the mean and the error bar represents ± standard deviation .

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Figure 7E-S. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E-H,Q) CDH5 protein, (I-L,R) CTNNB1 protein, (M-P,S) PRKCB protein. (E-P) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q-S) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


Figure 1 ( A Representative confocal images of HeLa and TRIM16KO cells treated with ( A ) MG132 ( 20 µM , 2h ) , scale bar : 7 . 5 µm . and the samples were processed for IF analysis with Ub and p62 antibody . ( B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( B ) MG132 ( 20 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . ( C The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm . D ) Representative confocal images of HeLa and TRIM16KO cells treated with ( D ) H2O2 ( 200 µM , 2h ) and the samples were processed for IF analysis with Ub and p62 antibody . E ) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( E ) H2O2 , ( 200 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . F ) The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm . ( G ) Western blot ( WB ) analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells treated with MG132 ( 50 µM , 1h ) and probed with indicated antibodies . ( H , I ) Densitometric analysis ( using ImageJ ) of WB normalized with α - Tubulin or Lamin - B1 . Data information : Unless otherwise stated , scale bar : 10 µm . ( J ) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 ( 20 µM , 2h ) and processed for IF analysis with Ub and LC3B antibody . ( K , L ) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with ( K ) Bafilomycin A1 ( 300 nM , 3h ) alone or ( L ) with MG132 ( 10 µM , 4h ) as indicated . L . E , Low exposure ; H . E , high exposure . ( M ) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 ( 200 µM , 2h ) and processed for IF analysis with Proteostat dye , Ub , and p62 antibody . To show the cellular details , the brightness of TRIM16KO cells images was increased more than control

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.(H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm.(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells


Figure 2 ( A ) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies . ( B ) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies . ( C ) Left panel , western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies . Right panel , densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) . D - G ( D , F ) HEK293T cells were either transiently transfected with an empty vector or a Flag - tagged - TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . ( E , G ) Densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . ( H ) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide ( 100 µM ) for the indicated period and probed with different antibodies as indicated . The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X - ray film . ( I ) Quantification of NRF2 , p62 , and KEAP1 band intensities relative to actin . n = 3 , * * p < 0 . 005 , * p < 0 . 05 ( Student ' s unpaired t - test ) . ( J ) Immunoprecipitation ( IP ) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 ( 10 µM , 4h ) . IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( L ) absence of MG132 ( 10 µM , 4h ) . IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( M ) presence of MG132 ( 10 µM , 4h ) . ( N ) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates . Mr , Molecular Weight Marker . ( O ) Co - IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates . ( P ) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates .

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Figure 2(A) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).D-G (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag-tagged-TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. (E, G) Densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, *p < 0.05 (Student's unpaired t-test).(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).(N) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, Molecular Weight Marker.(O) Co-IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.(P) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.


Figure 3 ( A , B ) Co - IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16 . ( C ) Co - IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16 . ( D ) Co - IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16 .

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Figure 3(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.(C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.(D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.


Figure 4 ( A - C ) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co - IP assays using transiently transfected plasmid constructs as indicated . Two different variants of ubiquitin protein are used , one that can only be ubiquitinated at Lysine 48 residue ( HA - K48 - UB ) and other that can be only ubiquitinated at Lysine 63 residue ( HA - K63 - UB ) . All other lysine residues are mutated . L . E , Low exposure ; H . E , High Exposure . Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays . Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays . ( F ) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co - IP assays using transiently transfected plasmid constructs as indicated . ( G ) Western blot analysis of lysates from HeLa , TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 ( 20 µM , 2h ) and the blot is probed with indicated antibodies . ( H ) Western blot analysis of lysates from HeLa , TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 ( 20 µM , 2h ) and the blot is probed with indicated antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.(F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated.(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


Figure 5 ( A ) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated . ( B ) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown . The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2 . L . E , Low exposure ; H . E , High Exposure . ( C ) Densitometric analysis of NRF2 band intensity normalized with actin . ( D ) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2 . 5 µM ( 2 h ) of As2O3 . ( E - G ) RNA isolated from HeLa and TRIM16KO cells , untreated or treated with MG132 ( 20 µM , 2h ) were subjected to qRT - PCR with primers of genes as indicated . The fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 , * * p < 0 . 005 ( Student ' s unpaired t - test ) . RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) . RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) . ( K ) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated . ( L ) Densitometric analysis of Ub smear intensity normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . ( M ) Representative confocal images of control , Ubb and Ube2n siRNA transfected cells treated with H2O2 ( 200 µM , 2h ) and IF was performed with Ub and p62 antibodies . Scale bar : 10 µm . ( N ) The graph shows the percentage of cells with protein aggregates . Data from ≥ 10 microscopic fields for each condition ( 40X ) , n = 2 , Mean ± SD . ( O ) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated .

O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 5(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.(C) Densitometric analysis of NRF2 band intensity normalized with actin.(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test).RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.(L) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.(N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD.(O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated.


Figure 6 ( A ) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies . ( B , C ) RNA isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected cells were subjected to qRT - PCR with primers of genes as indicated . The fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm . WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with MG132 ( 20 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel . Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel . ( H ) Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) and subjected to WB analysis with Myc , GFP and indicated antibodies . ( I ) Representative confocal images of Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) and subjected to IF with Ub and p62 antibodies . ( J ) Graph represent % of cells with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.


Figure 7WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 . WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 . ( E ) Confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm . ( F ) The graph shows the percentage of cells with TRIM16 - Ub or TRIM16 - Ub - p62 co - localized dots / aggregates . Data from ≥ 10 microscopic fields , n = 2 , mean ± SD . ( G ) Representative confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm . ( H ) Co - IP analysis of the interaction between two differently tagged TRIM16 variants . ( I ) WB analysis of detergent - soluble and - insoluble DSS - cross - linked fractions of HeLa and TRIM16KO cells , untreated or treated with MG132 ( 10 µM , 4h ) and probed with indicated antibodies . ( J ) Co - IP analysis of the interaction between Flag - tagged TRIM16 and Myc - tagged TRIM16 full length and its domain deletion proteins .

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Figure 7WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag-p62 probed with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.(E) Confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm. (F) The graph shows the percentage of cells with TRIM16-Ub or TRIM16-Ub-p62 co-localized dots/aggregates. Data from ≥ 10 microscopic fields, n=2, mean ±SD. (G) Representative confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm.(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.(I) WB analysis of detergent-soluble and -insoluble DSS-cross-linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 µM, 4h) and probed with indicated antibodies.(J) Co-IP analysis of the interaction between Flag-tagged TRIM16 and Myc-tagged TRIM16 full length and its domain deletion proteins.


Figure 8 ( A ) MTT assays performed at different time points with HeLa and TRIM16KO cells , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . ( B ) Images of HeLa and TRIM16KO cells , untreated or treated with As2O3 ( 2 . 5 µM , 4h ) . Scale bar : 400 µm . Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( C ) As2O3 ( 2 . 5 µM , 4h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( D ) H2O2 ( 400 µM , 2h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( E ) puromycin ( 5 µg / ml , 6h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( F ) MG132 ( 20 µM , 8h ) . ( G ) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Arrowheads indicate the cleaved form of PARP - 1 and Casapse - 3 .

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Figure 8(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h).(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.


Figure 9 ( A , B ) Clonogenic assays were performed with HeLa and TRIM16KO cells , untreated or treated with As2O3 or MG132 . ( C ) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points . As2O3 is injected when tumor volume of all groups was > 100mm3 ( day 0 in the graph ) . Tumor volumes were measured on an interval as indicated . Mean ± SE , n = 6 ( each group ) , * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( ANOVA ) . ( D ) Graph shows the % tumor regression ( Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100 ) . Mean ± SE , n = 6 ( each group ) , * * p < 0 . 005 ( Student ' s unpaired t - test ) . ( E ) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3 . ( F ) Representative pictures of dissected tumors . ( G ) Immunohistochemistry analysis performed with KI - 67 cell proliferation marker and hematoxylin nuclear stain . The graph represents the % of cells with KI - 67 staining . > 400 cells were counted for this analysis from three different sections from different animals , mean ± SD , * p < 0 . 05 ( Student ' s unpaired t - test ) . Scale bar : 50 µm . ( H ) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated . L . E , Low exposure ; H . E , High Exposure .

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Figure 9(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test). (E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors. (G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.(H) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure.


Figure 1A . IFT22 nucleotide - binding experiments . Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22 / 74342 - 401 / 81397 - 450 core complex incubated with mant - labeled GDP ( mant - GDP ) or non - hydrolysable GTP / ATP analogs ( mant - GMPPNP / mant - AMPPNP ) . The fluorescence intensity is plotted as a function of protein concentration . Data were fitted to a single - site binding equation for determination of the dissociation constant ( Kd ) . Kd values and standard deviations are calculated from three independent experiments . D . Top : Cartoon representation of IFT22 ( grey ) with positions of two nucleotide - binding mutants highlighted . GTP is shown in stick representation and Mg2 + as a ball . D175 ( blue ) is the unusual residue binding the guanine base while S19 ( pink ) is a conserved residue required for coordination of the Mg2 + cation and is commonly mutated to an asparagine to prevent nucleotide binding . Bottom : Nucleotide - binding experiments of IFT22 nucleotide - binding mutants D175E , D175A and S19N with fluorescently labeled nucleotides . Only the S19N mutation ( light pink ) abolished IFT22 nucleotide - binding ability completely .

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Figure 1A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.


Figure 3C . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged TbIFT22 ( WT and mutants ) and untagged CrIFT25 / 27 / 74 / 81 . WT TbIFT22 is able to pull down the Cr tetrameric complex , thus forming a chimeric IFT - B1 pentamer , while both the A86R and S19N mutant fail to bind the complex .

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Figure 3C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.


Figure 4A . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT74342 - 401 / 81397 - 450 and untagged IFT22 ( WT and mutants ) . Pulldowns were done from cell lysates of co - expressed proteins . Lanes 1 - 4 show similar total expression levels of the different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . The IFT22S19N ( inactive GTPase mutant ) and the IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with the IFT74342 - 401 / 81397 - 450 complex in the pull - down experiment . B . SDS - PAGE gel of a Ni2 + - NTA pulldown using the His - tagged IFT7479 - 401 / 811 - 450 and untagged IFT22 ( WT and mutants ) . Pulldowns were done from cell lysates of co - expressed proteins . Lanes 1 - 4 show similar total expression levels of the different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . The IFT22S19N ( inactive GTPase mutant ) and the IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with the IFT7479 - 401 / 811 - 450 complex in the pull - down experiment . C . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - length IFT25 / 27 / 74 / 81 complex with His - tagged IFT22 ( WT and S19N mutant ) . The nucleotide - binding deficient mutant IFT22S19N interacts with IFT25 / 27 / 74 / 81 in the pull - down experiment . D . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - length IFT25 / 27 / 74 / 81 complex with His - tagged IFT22 ( WT and A86R mutant ) . The IFT74 / 81 - binding mutant IFT22A86R fails to pull down the full - length tetrameric complex .

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Figure 4A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment.B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment.C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment.D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.


Figure 5A . Western blot analysis of the IFT22RNAi + GFP : : IFT22S19N cell line probed with the anti - IFT22 antibody ( bottom ) and with an anti - paraflagellar rod ( PFR ) as loading control ( top ) . B . IFA of the indicated trypanosome cell lines using the mAb25 ( marker for the axoneme , left panels ) and an anti - IFT172 antibody ( marker for IFT , right panels ) . The top panels show the phase contrast images merged with DAPI ( cyan ) that stains nuclear and mitochondrial DNA . Scale bars correspond to 5 µm . C . Kymographs showing the movement of the GFP : : IFT22S19N in the presence ( left ) or the absence ( right ) of the IFT22 endogenous protein . Note the improved signal - to - noise ratio in the absence of endogenous IFT22 . P and D mark the proximal and distal ends of the flagellum , respectively . Scale bars are 5 seconds ( time , vertical ) and 5 µm ( length , horizontal ) D . Dot plot representation of flagellum length in the indicated cell lines and conditions . For each condition , 100 flagella were measured and statistical analysis was performed with the ANOVA test . Significant differences are indicated with a star ( P < 0 .

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Figure 5A. Western blot analysis of the IFT22RNAi+GFP::IFT22S19N cell line probed with the anti-IFT22 antibody (bottom) and with an anti-paraflagellar rod (PFR) as loading control (top).B. IFA of the indicated trypanosome cell lines using the mAb25 (marker for the axoneme, left panels) and an anti-IFT172 antibody (marker for IFT, right panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. Scale bars correspond to 5 µm.C. Kymographs showing the movement of the GFP::IFT22S19N in the presence (left) or the absence (right) of the IFT22 endogenous protein. Note the improved signal-to-noise ratio in the absence of endogenous IFT22. P and D mark the proximal and distal ends of the flagellum, respectively. Scale bars are 5 seconds (time, vertical) and 5 µm (length, horizontal)D. Dot plot representation of flagellum length in the indicated cell lines and conditions. For each condition, 100 flagella were measured and statistical analysis was performed with the ANOVA test. Significant differences are indicated with a star (P<0.0001