EMBO/SourceData huggingface dataset:
Figure 4B RT-qPCR quantification of Srsf3 and Cdkn1a (p21) mRNA levels in Srsf3-knockout (KO) and control (Ctrl) iPSCs (*p<0.05, ***<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).C Quantification of Cdkn1a mRNA expression by RT-qPCR during reprogramming in Srsf3-KO and control cells (***p<0.001, ****p<0.0001, Two-way ANOVA, data as mean ± SEM, n=3, biological replicates).D RT-qPCR quantification of CDKN1A (p21) levels in HEK293T cells overexpressing empty vector, WT-miR-17-92, 17/20a-∆CNNC and total-CNNC∆ expression constructs (*p<0.05, **p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).E RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells over expressing empty vector, SRSF3-FL and SRSF3-∆RS constructs (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates). F RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells overexpressing scrambled control, miR-17, miR-20a or miR-17+miR-20a mimics (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).
Figure 5A Western blot analysis of SRSF3 and CDKN1A expression in SRSF3-T2A-GFP overexpressing (SRSF3) and control (GFP) LIM1215 cells. GAPDH served as a loading control.B TaqMan analysis of miR-17-92 miRNAs in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and GFP-only control (GFP) LIM1215 cells (*p<0.05, ****p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).C RT-qPCR quantification of CDKN1A (p21) mRNA levels in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells (**p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).D Left: Schematic of the Firefly luciferase reporter constructs Right: Luciferase activity in SRSF3 overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells transfected with the reporter constructs (**p<0.01, ***p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).F Quantification of fraction of cells in G1, S and G2 phases in SRSF3 overexpressing cells (SRSF3-GFP) relative to control (GFP) LIM1215 cells (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).
Figure 6A, B RT-qPCR quantification of SRSF3 (A) and CDKN1A (B) mRNA levels in colorectal tumours and their paired normal samples (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=25, biological replicates). N=normal, T=tumour.D Association between SRSF3 expression and tumour (T) stage. T1 (n=3), T2 (n=5), T3 (n=11) and T4 (n=3) tumours, SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max, n=biological replicates). E Association between SRSF3 expression and tumour differentiation status. Moderately (Mod.) differentiated tumours (n=19) and poorly differentiated tumours (n=5), SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max; n=biological replicates).G SRSF3 and CDKN1A expression in TCGA COAD data (version 2016_01_28; tumour n=459 and normal n=41 (biological replicates); RSEM, RNA-seq by Expectation Maximation; **p<0.01, two-tailed unpaired Student's t-test, central band=median, dotted lines =25th and 75th percentile).
Figure 1(B) Representative spinning disc confocal microscopy images of iDCs cultured in 2D suspension (left) or 3D collagen (middle) 8h post seeding. Scale bars = 20µm. Cells were stained with phalloidin-Atto390 to visualize F-actin and cell morphology. The dotted lines indicate which area are shown in higher magnification in the panel on the right. Scale bars = 10µm.Comparison of the volume (G) of iDCs derived from four donors (n = 1559 to n = 4675 each, mean +/-SD) cultured either in 2D suspension or 3D collagen and measured with RT-DC at a flow rate of 0.12 µl/s. A significant increase in deformability of iDCs when cultured in collagen was found , whereas no significant difference in cell volume (G) For statistical significance testing, linear mixed model analysis was performed to calculate ANOVA p-values; *p < 0.05, **p < 0.01, ***p < 0.001.(I-J) Two days post culturing in 2D suspension or 3D collagen, RNA was extracted and the transcriptome was determined by Clariom™ S Pico assay. The left panels show the heatmap related to the comparison of the expression between 2D suspension and 3D collagen profile of the untreated (mock, top) and LPS treated cells (bottom) (adjusted p-value < 0.05). Inside each cluster, two subclusters identify the 2D suspension and the 3D collagen cells. The right panels show the set of statistically enriched pathways identified with the genes in the heatmap using metascape.
Figure 2(C) Percentage of p24+ cells at 2 d.p.i. (left) and 4 d.p.i. (right). Graphs depict mean values +/- SD for cells from five donors. ** p<0.01 calculated with 2way ANOVA test.
Figure 3(B) Representative spinning disc confocal microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 h.p.i. Cells were stained with Phalloidin-Atto390 to visualize F-actin. Vpr-mRuby2 particles are shown in red. Scale bar = 20µm. Arrow heads highlight virus particles in 3D collagen. The dotted lines indicate which area are shown in higher magnification. Scale bars = 2µm (C) Vpr signal per cell. Graphs depict means values +/- SD for cells from four donors *** p< 0.001 calculated with 2way ANOVA test.Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (E) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. ** p<0.01calculated with 2way ANOVA test.Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (F-G) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. (left) and 48h p.i. (right) *p<0.05 calculated with 2way ANOVA test. The graphs show mean values +/- SD for cells from five and three donors, respectively.
Figure 4(B) Representative dot plots of FACS analysis to quantify the number of productively infected cells. Shown is the CD3-PerCp-Cy5-5 fluorescence intensity against intracellular p24-FITC after gating on live cells. The gate denotes the productive infected p24+ cell population with the percentage of p24+ CD4 T cells indicated. (C) Percentage of p24+ cells at 2 d.p.i. (top) and 4 d.p.i. (bottom). Graphs depict mean values +/- SD for cells from four donors.(F) Manual quantification of percentage of iDCs interacting with CD4 T cells over the 2 hour time course. Bars indicates mean value for cells from two donors.
Figure 5(C) Representative dot plots of FACS analysis to quantify the number of productively infected cells that upregulated CD86. Shown is CD86-PerCp-770 fluorescence intensity against intracellular p24-FITC. The gates indicate the percentage of p24+ and p24- cells together with the percentage of CD86+ and CD86- cell. (D) Ratio between the percentage of CD86 positive and CD86 negative cells for HIV-1 R5 infected iDC in the presence or absence of Vpx and for LPS treaded iDCs, as positive control. The graph shows mean values +/- SD for cells for five donors. n.s calculated with 2way ANOVA and Sidak's multiple comparison test.
Figure 6(C-E) Graphs show the fold change increase, over uninfected control, for infection with HIV-1 R5 in the presence of Vpx (C), HIV-1 R5 (D) or HIV-1 R5 in the presence of Vpx and Raltegravir control (E) of the 16 selected genes with antiviral properties for cells from three donors. Bars represent the mean +/- SD * p< 0.05 calculated with 2way ANOVA with Sidak test.
Figure 7(A-E) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen or 2D suspension for two days and the surface expression of CD4 (A), CCR5 (B), DC-SIGN (C), SIGLEC-1 (D), and MMR (E) was analyzed by FACS. Representative histograms of FACS analysis showing fluorescence intensity for each receptor analyzed (Top). The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample for cells from three to six donors (bottom) Bars represent mean +/- SD. *** p< 0.001; ** p<0.01 calculated with paired t-test.(F) Representative images showing sum slice z-projections. Images from the DC-SIGN channel of cells kept in 2D suspension or 3D collagen were identically processed and are shown with identical brightness/contrast settings. The actin signal was adjusted according to its brightness in each image individually to identify the cells. Scale bar = 20µm (G) The graph shows the fluorescence intensity mean of the DC-SIGN channel from cells grown in 2D suspension or 3D collagen. Bars define the mean +/- SD and dots represent individual cell analyzed. N of cells in 2D suspension = 34; N of cells in 3D collagen = 54. *** p< 0.001 calculated with Mann-Whitney test.iDCs were spin-transduced with VLPs carrying Vpxmac239 one day prior to infection. Cells were incubated with anti-DC-SIGN and anti-MMR blocking antibody or isotype control for 30 min, infected with HIV-1 R5 Vpr-Blam for 4 hours to asses fusion or for 48 hours to asses productive infection. (I) Percentage of positive cells for the presence of the CFF2 product at 4 hours p.i. (J) Percentage of p24+ cells at 48 hours p.i. The graphs show mean values +/- SD for cells from three donors. *p<0.05 calculated with 2way ANOVA test.
Figure 8(G) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen 2D collagen or 2D suspension for two days and the surface expression of DC-SIGN was analyzed by FACS. The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample indicated as fold change. Graphs represent the mean for cells from two donors.(H) iDC differentiated from peripheral blood monocytes for five days were spin-transduced with VLPs carrying Vpxmac239. One day later, iDCs were infected with HIV-1 R5 and cultured in 2D suspension, 2D collagen or 3D collagen. Productive infection was analyzed by p24 intracellular staining and FACS at 4 (d.p.i). The graph depicts mean values for cells from two donors.
Figure 1C CBB staining of purified p62 (268-440 aa), p62 (320-440 aa), SNAP-Atg1, and SNAP-ULK1.F In vitro kinase assay. Purified recombinant p62 (268-440 aa), p62 (320-440 aa), or mCherry-p62 was incubated for 20 min at 30 °C with purified SNAP-Atg1 or SNAP-ULK1 in the presence or absence of ATP. Reactions were then terminated by adding LDS sample buffer containing reducing agent, followed by immunoblot analysis with the indicated antibodies. As positive and negative controls, Huh-1 cell lysates treated with or without lambda protein phosphatase (λPP) were used. Data were obtained from three independent experiments. Asterisks show possible dimeric structures of p62 (268-440 aa), p62 (320-440 aa) and mCherry-p62.
Figure 2A In vitro formation of p62-8xUb condensates. 10 μM SNAP-8xUb labelled with Alexa Fluor 649 was mixed with 10 μM mCherry-p62 wild-type, mCherry-p62S403E S407E, or mCherry-p62S349E and observed by fluorescence microscopy. Scale bars: 20 μm. The graph indicates the quantified area of p62 condensates formed by wild-type or mutant p62. Data are means ± s.d of wild-type p62, p62S403E, S407E and p62S349E condensates (n = 4). Statistical analysis was performed by Šidák's test after one-way ANOVA.C Immunofluorescence microscopy. Wild-type, FIP200- or ULK1-knockout Huh-1 cells were immunostained with indicated antibodies. The mean fluorescence intensities of ULK1 on p62 bodies per cell were quantified in each genotype (n = 249 cells). Horizontal bars indicate medians, boxes the interquartile range (25th-75th percentiles) and whiskers 1.5× the interquartile range; outliers are plotted individually. Statistical analysis was performed by Šidák's test after one-way ANOVA. Scale bars, 10 μm (main panels), 1 μm (inset panels).D Immunofluorescence microscopy. Wild-type and FIP200-knockout Huh-1 cells were transfected with GFP-ULK1 or GFP-ULK2 and immunostained with anti-p62 antibody. The mean fluorescence intensities of ULK1 on p62 bodies in each cell were quantified for each genotype (n = 79 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).
Figure 3A Immunoblot analysis. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h, and the cell lysates were subjected to immunoblot analysis with indicated antibodies. The asterisk indicates non-specific bands. Data shown are representative of three separate experiments. Bar graphs show the results of quantitative densitometric analysis of Ser349- or Ser403-phosphorylated p62 forms relative to total p62 (n = 3), and of Ser318-phosphorylated ATG13 relative to total ATG13 (n = 3). Data are means ± s.e. Statistical analysis was performed by Welch's t-test.B Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S403) to p62 on p62 bodies and the size and number of p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).C Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S349) to p62 and the signal intensity of KEAP1 on p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).D Gene expression of NRF2 targets. Total RNAs were prepared from Huh-1 cells treated with or without 2.5 µM MRT68921 for 6 h. Values were normalized against the amount of mRNA in non-treated Huh-1 cells. qRT-PCR analyses were performed as technical replicates on each biological sample. Data are means ± s.e. Statistical analysis was performed by two-sided Welch's t-test.
Figure 4A Fluorescence microscopy. GFP-p62, GFP-p62S349E, GFP-p62S349A, or GFP-p62T350A were co-transfected with mCherry or mCherry-KEAP1 into Huh1 p62 KEAP1 double-knockout cells. Twenty-four hours after transfection, the fluorescence images were observed.E FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. The half-time of recovery (t50) and mobile fraction (MF) of mCherry-KEAP1 was measured by FRAP of whole p62 bodies (n = 7). Data are means ± s.d. Statistical analysis was performed by two-sided Welch's t-test.F FLIP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. The fluorescence loss of mCherry-KEAP1 in GFP-p62 bodies (n = 14) was measured 30 min after photobleaching over a large area of cells.G FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. t50 and MF of mCherry-KEAP1 were measured by FRAP of the central portions of p62 bodies (n = 10). Data are means ± s.d. Statistical analysis was performed by two-sided Welch's t-test.
Figure 5D RNA-seq analysis of livers of p62+/+ and p62S351E/+ mice at P19 (n = 3). Volcano plots of differentially expressed genes in p62+/+ versus p62S351E/+ mice (red points: FDR < 0.05 and log2FC > 1, blue points: FDR < 0.05 and log2FC < -1). Green points are the targets of transcription factor NRF2.F Immunoblot analysis of p62+/+ (n = 7) and p62S351E/+ (n = 3) mice at P19. Liver homogenates were subjected to immunoblot analysis with the indicated antibodies. Bar graphs show the results of quantitative densitometric analysis. Data are means ± s.e. Statistical analysis was performed by Welch's t-test. Note that both faster and slower migrating bands in the KEAP1 blot represent translational products from the Keap1 gene.G, H Hematoxylin and eosin (HE) staining (G) and immunohistochemical analysis of NQO1 (H) of livers from p62+/+ and p62S351E/+ mice at P19. Scale bars, 100 μm (low magnification panels), and 10 μm (high magnification panels).i Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, total cholesterol, blood urea nitrogen (BUN), and creatinine from p62+/+ (n = 4 at P12, n = 10 at P15) and p62S351E/+ (n = 4) mice at P12 and P15 were measured. IU/l, international units/liter. Data are means ± s.e. Statistical analysis was performed by Tukey's test after one-way ANOVA.
Figure 1(A and B) qPCR analysis (n = 3) of BRCA1 mRNA (A) and pre-mRNA levels (B) in different cell cycle phases of HeLa cells.(C) ChIP analysis (n = 3) of Pol II binding to BRCA1 promoter during G1 and S phases in HeLa cells. (D) ChIP analysis (n = 3) of Pol II-pSer2 binding to BRCA1 3'UTR during G1 and S phases in HeLa cells. Pol II-pSer2, phosphorylation of RNA Pol II CTD Ser2.(G and H) HeLa cells were synchronized to G1 or S phase, then the degradation rates of endogenous (G) and exogenous (H) BRCA1 were analyzed by western blot. The quantification of BRCA1 relative levels according to GAPDH level are labeled below the BRCA1 band. CHX, Cycloheximide that inhibits nascent protein production. SFB-BRCA1, exogenous BRCA1 with flag tag in N-terminal.Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1(n = 3) (O) or BRCA1 exon5 alternative splicing (n = 3) (AS) during the indicated cell cycle phases (P) by semi-quantitative RT-PCR (sqRT-PCR). Left, bar plot showing the concentration of BRCA1 isoforms in exon5 AS (O) or the ratio of BRCA1 WT isoform in exon5/11 AS (P the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. (P) HeLa cells were transfected with UPF1 siRNA, then BRCA1 WT and 5q isoform mRNA levels during the indicated cell cycle phases were analyzed by qPCR (n = 3). siNC, transfected with control siRNA. siUPF1, transfected with UPF1 siRNA.Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1(n = 3) during the indicated cell cycle phases by semi-quantitative RT-PCR (sqRT-PCR). siNC, transfected with control siRNA. siUPF1, transfected with UPF1 siRNA.
Figure 2(C and D) RNA pull-down assay shows RBM10 binding to BRCA1 exon5 (C) and exon11 (D) RNA in vitro.(G and H) The BRCA1 protein (G) and mRNA levels (n = 3) (H) in 293T and RBM10-KD cells.(N and O) qPCR analysis (n = 3) of BRCA1 WT isoform mRNA level in exon11 AS during the indicated cell phases after depleting RBM10 in HeLa (N) and MCF-7 (O) cells.(P) Results of western blot analyzing BRCA1 levels in HeLa and RBM10-KD cells in the indicated cell cycle phases.
Figure 3(A to D) Analysis of BRCA1 exon5 (A) and exon11 (C) AS by by semi-quantitative RT-PCR (sqRT-PCR) (n = 3), and analysis of BRCA1 exon5 (n = 3) (B) or exon11 (n = 3) (D) AS by minigene splicing reporter system after the depletion of RBM10. Bar plot shows the ratio of BRCA1 WT isoform in exon5/11 AS, the figure above is the representative DNA gel, the right is signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units.(F) qPCR (n = 3) analyzing the WT and D11q isoform levels (exon11 AS) after RBM10 overexpression in 293T cells.
Figure 4(A and B) Western blot analysis of RBM10 phosphorylation in the indicated cell phases (A) or G1/S phase (B). In (B), cells were treated with the indicated CDK inhibitors (10 μM) for 6 h. CDK1i, Cucurbitacin E. CDK2i, SB1317. CDK4/6i, Palbociclib.(F) Analysis of BRCA1 exon5 AS by by sqRT-PCR (n = 3) in in G1/S phase of 293T cells treated with CDK2i. In (F) bar plot showing the ratio of BRCA1 WT isoform in exon5 AS, the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. Data are presented as mean ± s.d. in all qPCR.(I) Mass spectrometry analysis showing RBM10 phosphorylation at S89 site.(N) Analysis of BRCA1 exon5 AS by sqRT-PCR (n = 3), 293T cell was transfected with RBM10 S89D variant in G1 phase. In (N), bar plot showing the ratio of BRCA1 WT isoform in exon5 AS, the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. Data are presented as mean ± s.d. in all qPCR.(O to Q) Phosphorylation at S89 of the indicated RBM10 variants (O), WT RBM10 in the indicated cell cycle phases (P), and WT/S89A mutant RBM10 after CDK2i treatment in G1/S phase (Q) were analyzed in 293T cells.
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.
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).