Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: a , Schematic description of the dualPIM system. b , IEM analyses of dualPIM aggregates induced for 30 min, 2 h or 6 h. Cryosections were labelled individually with anti-GFP (30 min and 2 h time points) or in combination with anti-LAMP2 antibodies (6 h time point). Insets are shown below the overviews. Scale bars, 500 nm (overviews) and 250 nm (insets). c , HeLa cells transiently expressing dualPIM were imaged until 16 h after triggering aggregation with rapa2 treatment. Time-lapse images showing multiple fragmentation (white arrows) and degradation (white arrowhead) events are presented in the panel. See also Supplementary Video and Extended Data Fig. . CM, control medium. d , DualPIM cells were treated ± 40 µM VER for 19 h. e , f , Quantification of the average dualPIM puncta size at 19 h ( e ) and degradation rates ( f ). P = 0.0055 ( e ); P < 0.0001 ( f ). g , DualPIM cells were transfected with the indicated siRNAs (also Supplementary Table ) and imaged for 24 h. Representative images at 24 h are shown. h , i , Quantification of dualPIM puncta average size at 24 h ( P values versus siCtr: P = 0.001 (siCtr + BAF); P = 0.024 (siHSP70/siHSPA1A); P < 0.001 (siHSP110); P < 0.001 (siDNAJA2); P < 0.001 (siDNAJB6)) ( h ) and degradation rates ( P values versus siCtr: P < 0.001 (siCtr + BAF); P = 0.002 (siHSP70/siHSPA1A); P < 0.001 (siHSP110); P = 0.004 (siBAG3); P < 0.001 (siDNAJB6)) ( i ) of the experiment are shown. Images in c were acquired using the Nikon Eclipse microscope and images from d and g using the IncuCyte S3 system. Data are presented as mean ± s.d. Error bars represent the s.d. of three ( e , f , h , i , siHSPB1, siHSPB7, siHSP110, siDNAJA1, siDNAJA2, siDNAJB1 and siDNAJB4 condition), four ( h , i , siHSPBP1, siHSP70/siHSPA1A, siBAG3, siDNAJB2 and siDNAJB6 condition), eight ( h , i , siCtr condition) or ten ( h , i , siCtr + BAF condition) independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001. P values were calculated by a two-tailed t -test with Bonferroni correction ( e , f ) or one-way analysis of variance (ANOVA) following Dunnett’s post hoc test ( h , i ). Scale bars, 20 µm, except in a (5 µm).

Article Snippet: Detection of Lamp2 was performed using an anti-Lamp2 antibody (BD biosciences, 555803) follow by a rabbit anti-mouse (Rockland, 610-4120) and protein A-conjugated gold (GFP: 15 nm; LAMP2: 10 nm).

Techniques: Expressing, Control, Transfection, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a Volcano plot of differentially expressed core splicing factors ( n = 134) between the TCGA-OV cohort ( n = 374) and normal tissue in GTEx datasets ( n = 180). |log2FC| > 1 and an adjusted p value < 0.05 were considered significant. b , c Forrest plot of the hazard ratio for the association between 13 dysregulated splicing factors and overall survival ( n = 1023) and progression-free survival ( n = 1001) in patients with SOC from the Kaplan–Meier Plotter database. The cohort of patients with SOC was split by auto-select cutoff. Prognostic splicing factors (red) with a p < 0.05 were considered statistically significant. d , e MTT assay and flow cytometry determined the HEYA8 cell viability and apoptosis cell percentage after siBUD31, siSF3B1, siSF3B4, siSRSF4, siMATR3, and siCHERP treatment for 72 h ( n = 3 biologically independent experiments). * p < 0.05, ** p < 0.01. f BUD31 mRNA expression was analyzed in the TCGA-OV cohort ( n = 374) and normal tissue in GTEx datasets ( n = 180). g BUD31 protein level was analyzed in SOCs ( n = 93) and FTs ( n = 25) from the CPTAC dataset. SOC samples were classified into Stage 1 ( n = 2), Stage 3 ( n = 75), and Stage 4 ( n = 16) according to individual cancer stage. The bounds of the box were the upper and lower quartile with the median value in the center. The whiskers indicated the minima and maxima. h Statistical analysis of BUD31 expression from IHC staining of the tissue microarray containing 149 samples of SOCs and 73 samples of FTs. i Representative images of IHC staining with high, medium, and low BUD31 expression in our tissue microarray. j , k Kaplan–Meier analysis of the correlation between BUD31 expression and overall survival and progression-free survival of ovarian cancer patients based on data from our tissue microarray. The 95% confidence interval was shown as dotted lines. l , m Kaplan–Meier analysis of the correlation between BUD31 expression and overall survival and progression-free survival of ovarian cancer patients based on the Kaplan–Meier Plotter cohort. The p value was obtained by log-rank test ( b , c , j , k , l , m ), two-tailed unpaired Student’s t -test ( d , e , f , g ), and Chi-square test ( h ). Data are presented as means ± SD unless otherwise stated.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: MTT Assay, Flow Cytometry, Expressing, Immunohistochemistry, Microarray, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: GO biological process enrichment ( a ) and Circos plot ( b ) analysis were conducted on DEGs in the RNA-seq data from HEYA8 cells after BUD31 knockdown. c GSEA analysis was performed with the gene expression profile after BUD31 knockdown (Normalized ES = –1.222, p < 0.001). d , e Apoptotic cells were detected by flow cytometry after staining with Annexin V-PE/7-AAD in ovarian cancer cells after BUD31 knockdown with two strands of siRNAs (HEYA8 and OV90) or overexpression (HEYA8 and A2780) ( n = 3 biologically independent experiments). Apoptotic cells percentage included early and late apoptotic cells. Cells overexpressing BUD31 were treated with H 2 O 2 with a final concentration of 400 μM for 4 h before apoptosis detection. f Apoptotic markers were measured by western blot. BUD31 was knocked down in HEYA8 and OV90 cells and was overexpressed HEYA8 and A2780 cells in the presence of H 2 O 2 . The p value was obtained by two-tailed unpaired Student’s t -test ( e , f ), and data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: RNA Sequencing, Knockdown, Gene Expression, Flow Cytometry, Staining, Over Expression, Concentration Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: The EdU assay ( a ) and cell proliferation assay ( b ) were performed in ovarian cancer cells with BUD31 overexpression (HEYA8, A2780) or knockdown (HEYA8, OV90) compared to corresponding controls ( n = 3 for the EdU and n = 5 biologically independent experiments for the cell proliferation assay). Cell proliferation was measured using the MTT cell proliferation assay. Absorbance at 570 nm at each time point was compared to the initial state (time = 1 day). c Luciferase signals of intraperitoneal injected nude mice and photon flux quantification. Nude mice were injected with luciferase-expressing HEYA8 cells with a dox-inducible BUD31 knockdown system ( n = 6 per group). Administration of doxycycline (1.2 g/L) started one week after the cell implantation. The bounds of the box were the upper and lower quartile with the median value in the center. The whiskers indicated the minima and maxima. d IHC staining of BUD31 and Ki-67 expression in xenograft tumors of HEYA8 cells with BUD31 knockdown compared to corresponding controls. e TUNEL assay to quantify the apoptotic cells in xenograft tumors with BUD31 knockdown compared to corresponding controls. The p value was obtained by two-tailed unpaired ( a – c ), and the results are presented as the mean ± SD. * p < 0.05, ** p < 0.01. Source data are provided as a Source Data file.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: EdU Assay, Proliferation Assay, Over Expression, Knockdown, MTT Cell Proliferation, Luciferase, Injection, Expressing, Immunohistochemistry, TUNEL Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a GO biological process enrichment analysis of BUD31-interacting proteins in HEYA8 cells using immunoprecipitation coupled to mass spectrometry. b The correlation network between BUD31 and splicing factors was constructed in Cytoscape. Proteins belonging to the spliceosome were classified into PRP19 complex, SR protein, U1 snRNP, U2 snRNP, U5 snRNP, hnRNP protein, tri-snRNP, and others. c Immunofluorescence assays showed the co-localization of BUD31 (red) with splicing factor SC35 (green) in punctate nuclear speckles. The quantification and analysis of co-localization was performed with Coloc 2 and Plot Profile (Pearson’s r = 0.57, Manders’ M1 = 0.81, Manders’ M2 = 0.96). d High-density scatter plot of the SpyCLIP and Input reads counts aligned to the BUD31-binding regions. e Position of the SpyCLIP crosslinking regions relative to the crosslinking sites identified by PURECLIP. The SpyCLIP (deep blue) and input (gray) signals are shown around the crosslink sites. f Length distribution of the BUD31-binding regions and the crosslinking sites included in the corresponding binding regions. g Distribution of SpyCLIP crosslinking regions annotated by HOMER on genome elements. Processed regions were longer than 3 nucleotides. h SpyCLIP read distribution compared with input on the genome elements, including 5′ UTR, intron, exon, and 3′ UTR. i De novo motif analysis of BUD31 SpyCLIP clusters and statistical results of the top-four BUD31-binding motifs ranked by HOMER calculated p value. j Enriched sequence elements of the top-four BUD31-binding motifs. k Upset plot of the distribution of motifs 1–4 in the BUD31-binding regions. l BUD31-binding motif distribution in the exon region and the 300 bp flanking the 3ss or 5ss intron-exon junction site. The exon region was scaled such that the length was equal to 300 bp to normalize different exon lengths. m SpyCLIP reads intensity distributed around the alternative exons. Enhanced exons (deep blue) were included, and silenced exons (blue) were excluded after silencing BUD31. Randomly chosen exons were used as controls (yellow).

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: Immunoprecipitation, Mass Spectrometry, Construct, Immunofluorescence, Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a , b Pie chart depicting the proportions of different types of AS events in the RNA-seq data from HEYA8 cells after BUD31 knockdown or overexpression. SE skipped exons, RI retained introns, A5SS alternative 5′ splice site, A3SS alternative 3′ splice site, MXE mutually exclusive exons. c , d The proportions of genes (95% confidence interval) with different types of AS induced isoform switches after BUD31 knockdown ( n = 5651) and overexpression ( n = 2723). e Density plot of the long coding sequence isoforms (750–1750 bp) length distribution in HEYA8 cells with BUD31 knockdown compared with corresponding controls. Arrow, statistically significant regions. f Cumulative distribution function plot of coding sequence lengths of all annotated genes after BUD31 knockdown. g The differential expression of genes with increased NMD-sensitive isoforms fraction (dIF > 10%, n = 75) and decreased fraction (dIF < −10%, n = 36). The log 2 transformed fold changes were shown in boxplot (10-90 percentile). h Pathway enrichment of the NMD-sensitive and downregulated targets. i Venn diagram of 8780 BUD31-binding genes from the SpyCLIP-seq, 2472 genes from RIP-seq, and 2465 genes with AS events. The AS pattern and BUD31-binding sites in E2F4 ( j ), CDK16 ( m ) and RBCK1 ( o ) were visualized with IGV using the RNA-seq and SpyCLIP-seq data. The light blue region highlights the AS region and the BUD31-binding sites. Semi-quantitative RT-PCR was performed to validate the AS events in E2F4 ( k ), CDK16 ( n ), and RBCK1 ( p ) in HEYA8 cells with BUD31 knockdown compared with controls. l The protein expression of E2F4 and CDK16 was determined by western blot in HEYA8 cells knocking down BUD31. q , r MTT assay and flow cytometry were performed to determine the cell viability and apoptosis cells percentage after siBCL2L12 and siRBCK1 treatment. The p value was obtained by two-tailed unpaired Kolmogorov–Smirnov test ( f ) and Student’s t -test ( g , q , r ), and the results are presented as the mean ± SD.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: RNA Sequencing, Knockdown, Over Expression, Sequencing, Quantitative Proteomics, Transformation Assay, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, MTT Assay, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a The AS pattern and BUD31 direct binding sites in BCL2L12 were visualized with IGV using RNA-seq, RIP-seq, and SpyCLIP-seq data. The light blue region highlights the alternative exon and the BUD31-binding sites. b The Sashimi plots of exon 3 skipping in BCL2L12 in HEYA8 cells with BUD31 knockdown (red) and corresponding controls (blue). c , d Semi-quantitative RT-PCR and fragment analysis were performed to validate AS events in BCL2L12 after BUD31 knockdown or overexpression. e The relative expression ratio of BCL2L12 -L/ BCL2L12 -S was analyzed in HEYA8 ovarian cancer cells with BUD31 knockdown or overexpression ( n = 3 biologically independent experiments). f Schematic structure of two BCL2L12 transcripts. BCL2L12 -L is the full-length transcript, and BCL2L12 -S is a short transcript lacking exon 3 skipping, which generates a premature termination codon (PTC). g BCL2L12 -S expression was measured by qPCR in UPF1 knockdown and control HEYA8 cells treated with 10 μg/ml actinomycin D at the indicated times. h RIP-qPCR was performed to validate the interaction between BUD31 and BCL2L12 RNA in HEYA8 cells with the anti-BUD31 antibody ( n = 3 biologically independent experiments). * p < 0.05, ** p < 0.01. i RNA pull-down assay showed the interaction between BCL2L12 pre-mRNA and BUD31 protein. Protein expression was quantified and normalized with the Input sample. j RNA EMSA showed the binding of recombinant BUD31 and BCL2L12 pre-mRNA fragments. The upper band shows the complex of BUD31 protein and BCL2L12 pre-mRNA. k Relative BCL2L12 -L/S transcript expression was measured by qPCR in SOC samples ( n = 8) and FTs ( n = 4). l Isoform percentage of BCL2L12 -L and BCL2L12 -S in SOCs and normal ovaries from the TCGA-OV and GTEx datasets. m Correlation between BUD31 mRNA expression and the PSI value of AS events in the TCGA-OV dataset. P values and r values were calculated by Pearson’s correlation. n Kaplan–Meier analyzes the correlation between BCL2L12 exon 3 expression and overall survival based on TCGA data. The p values were obtained by two-tailed unpaired Student’s t -test ( e, g, h ) or log-rank test ( n ), and the results are presented as the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: Binding Assay, RNA Sequencing, Knockdown, Quantitative RT-PCR, Over Expression, Expressing, Control, Pull Down Assay, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a Correlation analysis of mRNA expression between BUD31 and BCL2L12 in SOCs and normal ovaries from the TCGA-OV and GTEx datasets. (Pearson’s r = 0.39). b Western blot analysis of BCL2L12 protein expression in HEYA8 cells with BUD31 knockdown or overexpression in A2780 cells. Protein expression was quantified and normalized with the control group. c Immunoblot analysis of BUD31 and BCL2L12 protein levels in HEYA8 cells transfected with shBUD31 or BCL2L12 overexpression vector. Apoptosis ( d ), clonogenic ( e ), and EdU ( f ) assays for investigating the potential of BCL2L12 to rescue the loss of BUD31 in terms of apoptosis and proliferation. g – i Xenograft experiments by subcutaneous injection were conducted in HEYA8 cells with dox-inducible BUD31 knockdown or BCL2L12 overexpression vector. Representative image ( g ), volume curves ( h ), and weight ( i ) of xenograft tumors showed that BCL2L12 could partially rescue the inhibitory effect of BUD31 on tumor growth ( n = 10). j BCL2L12 expression was analyzed in SOCs from TCGA-OV ( n = 374) and in normal ovaries from GTEx ( n = 180). k BCL2L12 mRNA expression was determined by qPCR in SOC ( n = 23) and FT ( n = 9) samples. l Kaplan–Meier analysis of BCL2L12 expression on the overall survival of ovarian cancer patients based on cohorts from Kaplan–Meier Plotter. The high and low-expression groups were separated based on the auto-select cutoff. All functional experiments were performed with n = 3 biological repeats. The p values were determined by a two-tailed unpaired Student’s t -test ( d , e , f , h , i , j , k ), or log-rank test ( l ), and the results are presented as the mean ± SD. * p < 0.05, ** p < 0.01. Source data are provided as a Source Data file.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Control, Transfection, Plasmid Preparation, Injection, Functional Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

doi: 10.1038/s41467-022-34042-w

Figure Lengend Snippet: a Schematic diagram of ASO target sites on BCL2L12 based on the BUD31-binding region on BCL2L12 as determined from the CLIP-seq data. b RT-PCR analysis of the BCL2L12 AS pattern in response to ASOs. c Western blot analysis of the BCL2L12 protein level in A2780 cells transfected with ASOs. Semi-quantitative RT-PCR analysis of the BCL2L12 AS pattern in HEYA8 ( d ) and A2780 ( e ) cells transfected with ASO2. Dose-dependence curve of ASO2-treated HEYA8 and A2780 cells showing increased skipping of exon 3 [(exon 3 skipped/exon 3 skipped + full-length) × 100] in relation to the log of the dose. The EC 50 was calculated in HEYA8 (63.77 nM) and A2780 (52.91 nM) cells. BCL2L12 and cleaved-caspase-3 were measured by western blot in A2780 cells treated with 200 nM ASO2 for different times (0, 24, 48, and 72 h) ( f ) and with different concentrations (0, 25, 50, 100, 200 nM) of ASO2 for 72 h ( g ). h Apoptotic cells were detected by flow cytometry after staining with Annexin V/7-AAD in A2780 and HEYA8 cells treated with ASO2 (200 nM). i , j EdU assay was performed in ovarian cancer cell lines treated with 200 nM ASO2. The IC 50 was calculated based on the MTT assay. k – n ASO2 intratumoral injection to subcutaneous tumor xenografts using HEYA8 cells. The xenograft model showed the inhibitory effect of ASO2 on tumor growth ( n = 6 mice per group) ( k ). The tumor weight ( l ) and volume ( m ) were measured for each group. The BCL2L12 and Ki-67 expression levels were evaluated with immunohistochemical staining, and the apoptosis level was determined by a TUNEL assay ( n ). All functional experiments were conducted with n = 3 biological repeats. The p values were obtained by two-tailed unpaired Student’s t test ( h , i , l , m ), and the results are presented as the mean ± SD. * p < 0.05, ** p < 0.01. Source data are provided as a Source Data file.

Article Snippet: Whole-cell extracts were incubated with 5 μg BUD31 antibody (Proteintech) overnight for 1 h at 4 °C, then incubated with magnetic Protein A/G beads (Millipore) for 2 h at 4 °C.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Staining, EdU Assay, MTT Assay, Injection, Expressing, Immunohistochemical staining, TUNEL Assay, Functional Assay, Two Tailed Test