Fig 2: TRPC3 controls senescence.a Cumulative population doublings (PD) of HPrFs either stably expressing doxycycline-inducible shRNA against TRPC3 or treated with siTRPC3 and their siControls (siCTL, scrambled sequences). Results are presented as mean ± SEM; n = 3). b EdU staining on MRC5 cells treated 10 days with siCTL or siTRPC3. % of EdU positive cells is shown (mean ± SEM; n = 3). Scale bar = 150 µm. ***P < 0.001 (Student’s t test, two-sided). c Effect of siRNA-mediated TRPC3 knockdown in HPrFs on: (Top) the cell structural parameters revealed with flow cytometry analysis—cell size (forward scatter; FSC) and internal complexity (side scattered, SSC), (Middle) cell growth reported by crystal violet staining and (Bottom) ß-gal activity revealed with SA-ß-gal staining (% of ß-galactosidase positive cells is shown, mean ± SEM). Readings were performed 10 days after TRPC3 knockdown. n = 3 biologically independent experiments. d p53 and p16 protein levels are shown for cells treated as in (c). Data are expressed as mean ± SEM (n = 3). Western blot images are representative of three independent experiments. e yH2AX staining and fluorescence microscopy of MRC5 cells treated as in (c) showing signs of DNA damage after 10 days of TRPC3 knock-down. % of yH2AX positive cells is shown (mean ± SEM, n = 3 biologically independent experiments). ***P < 0.001 (Student’s t test, two-sided). f Effect of TRPC3 overexpression in OIS. (Top) colony formation assay reported by crystal violet staining and (bottom) SA-ß-gal activity revealed with SA-ß-gal staining (% of ß-galactosidase positive cells is shown, mean ± SD). Readings were performed from ER:hRAS-MRC5 cells (bearing doxycycline-inducible TRPC3) in which senescence was induced by 4-OHT treatment over 10 days. To overexpress TRPC3, the cells were kept in doxycycline-containing medium for 3 weeks after senescence induction. g Cyclin D1, PCNA1 and p16 protein levels are shown for cells treated as in (f) and normalised against Calnexin expression used as loading control. Western blot images are representative of three independent experiments. Scale bar = 100 µm.

Fig 3: RNA Profiling for miR-1468-3p Target Genes in hCFshCFs were treated with antimiR-Ctrl for 24 h and treated with TGF-ß1 (5 ng/mL) for 24 h where indicated. At the end of the experiment, RNA was collected for RNA sequencing analysis, qPCR analysis, or WB analysis. Shown are (A) Gene Ontology (GO) enrichment analysis and (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for the genes downregulated by antimiR-1468-3p. (C) Heatmap representation of genes that were significantly regulated by antimiR-1468-3p in TGF-ß1-treated cells. (D) qPCR analysis of selected genes modulated by TGF-ß1 and/or antimiR-1468-3p. (E) WB and densitometry analysis (right panel) for p16 and p21. GAPDH was used as a loading control. qPCR, N = 3 per group. Data are expressed as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001 versus antimiR-Ctrl; #p < 0.05, ##p < 0.01, ###p < 0.001 versus antimiR-Ctrl+TGF-ß1.

Fig 4: Cardiac miR-1468-3p Expression Is Increased in Aged SCD Victims and It Promotes Senescence(A and B) Analysis for cardiac miR-1468-3p (A and B) and miR-1468-5p (B) expression in control subjects (control) and victims of sudden cardiac death (SCD) with primary myocardial fibrosis (PMF case). All samples were paired with matched age. The data are shown as relative to respective age control of miR-1468-3p. (C and D) Human cardiac fibroblasts were treated with 20 nM of either mimic-1468-3p or control sequence (mimic-Ctrl) for 24 h. (C) Quantitative analysis of senescence-associated ß-galactosidase (SA-ß-GAL) activity and representative staining. Scale bar: 100 µm. N = 4. (D) Western blot (WB) analysis and densitometry analysis for expression of p16, p21, and p53. Vinculin was used as a loading control. (E) Analysis for miR-1468-3p and miR-1468-5p expression in healthy control hearts at different ages. (F) Analysis for miR-1468-3p expression in young healthy control hearts and in hearts of 45- to 65-year-old SCD victims with PMF (PMF case). Data are expressed as mean ± SD. Student’s t test was used. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: GSC lines show differential sensitivities to PRMT5 inhibition.a Area above the curve (AAC) calculated from dose–response assays across 46 patient-derived GSC lines for GSK591 (red) and LLY-283 (blue) over a range of compound concentrations from 3 nM to 30 µM. DMSO was used as a control. A higher AAC represents greater sensitivity. Asterisk (*) denotes lines studied in more detail in this paper. b Common genomic alterations (derived from whole-genome sequencing data), found in 46 patient-derived GSC lines alongside the TCGA classification for each line (in the order of decreasing GSEA enrichment). GSC lines are ordered as in a. c Western blots for MTAP, CDKN2A, and GAPDH expression across the panel of GSC lines (ordered by increasing response to PRMT5 inhibitors and roughly aligned with b). The full GSC line panel experiment was run once, albeit a subset of GSC lines were run twice with similar results. d Quantification of senescence-associated ß-galactosidase-positive cells in four representative GSC lines. Cells were treated with 1 µM of the PRMT5 inhibitors, GSK591 and LLY-283, and controls, SGC2096 and DMSO, for 5 days. n = 3, mean ± SD. Two-tailed unpaired t test with Welch’s correction. p values: G411-GSK/SGC = 0.0079, G411-LLY/DMSO = 0.0035; G583-GSK/SGC = 0.1052, G583-LLY/DMSO = 0.0183; G729-GSK/SGC = 0.0071, G729-LLY/DMSO = 0.0076; G797-GSK/SGC = 0.0735, G797-LLY/DMSO = 0.0216. e Quantification of Annexin V+ cells in four GSC lines treated with 1 µM of the PRMT5 inhibitors, GSK591 and LLY-283, and controls, SGC2096 and DMSO, for 9–12 days (until cells of DMSO control were confluent). Data shown are representative of two independent experiments. f Cell numbers in GSC lines, G561, G583, G837, and G411 treated for 14 days with PRMT5 inhibitors, GSK591 and LLY283, after which drug was either washed out or left on and followed for another 14 days. Dashed line depicts the 14-day point after which the drug was washed out. Data shown are representative of two independent experiments. *p < 0.05; **p < 0.01. See also Supplementary Fig. 2. Source data are provided as a Source data file.