Fig 1: CSB depletion by histone H3 hypoacetylation of its promoter. a Quantitative RT-qPCR of DNMT3A, DNMT3B, and DNMT1. b DNA methylation (percent) of eight individual CpG sites in the CSB promoter at the indicated PN (positive control [CTL + ], a universal methylated (human) DNA standard). n = 3 independent experiments, mean ± SD; two-way ANOVA (F = 2.983, DFn = 5, DFd = 96, p = 0.0151) with post-hoc Tukey’s test. c Immunoblots of H3 acetylated (H3Ac) and total histone H3 (reprobed after H3Ac stripping) at the indicated PN, with GAPDH as a loading control. d Quantitative RT-qPCR of HDAC1 and HDAC2. e Quantitative PCR of DNA in a ChIP assay with either a-H3 acetylated or total a-H3 (material not available at PN35). Primers detect the occupancy of H3Ac/H3 at a specific region of the CSB promoter. Results are expressed as the percentage of input DNA normalized to H3 occupancy. f Scheme indicating the anacardic acid (AA) target. g Quantitative RT-qPCR of CSB upon treatment with increasing concentrations of AA or DMSO (Vehicle) for 24 h, and in untreated control (Untreated). h Immunoblot of H3 acetylated, total H3 histone, CSB, HTRA3, HTRA2, and p21 from whole-cell extracts with increasing concentrations of AA or DMSO (Vehicle) for 24 h, and 24 h after AA withdrawal and in the corresponding controls (Untreated). Samples on the same blot are framed; each frame displays the respective GAPDH used as a loading control (GAPDH (F-C staining in the middle blot)). Quantitative PCRs: n = 3 independent experiments; mean ± SD; one-way ANOVA (a DNMT1: F = 9.129, DFn = 5, DFd = 12, p = 0.0009; DNMT3A: F = 0.05094, Dfn = 5, DFd = 12, p = 0.9980; DNMT3B: F = 0.5325, DFn = 5, DFd = 12, p = 0.7481; d HDAC1: F = 12.93, DFn = 5, DFd = 12, p = 0.0002; HDAC2: F = 3.826, DFn = 5, DFd = 12, p = 0.0264; e H3: F = 2.298, DFn = 4, DFd = 10, p = 0.1304; H3Ac/H3: F = 5.064, DFn = 4, DFd = 10, p = 0.0171; g F = 9.516, DFn = 4, DFd = 10, p = 0.0019) with post-hoc Tukey’s test vs. PN16 (or Vehicle, g) when not specifically indicated. i Quantitative PCR analysis of a DNA fragment in the CSB promoter from ChIP assay with a-H3 acetylated in the presence and in the absence of AA treatment; n = 3 independent experiments, mean ± SD; unpaired Student’s t-test (two-tailed) (t = 3.908, DF = 4) vs. Vehicle. Source data are provided as Source Data files.
Fig 2: CSB knockdown induces premature p21-dependent senescence. a Scheme of the experiment. b Immunoblot of CSB at PN20. GAPDH was used as a loading control. c Quantitative RT-qPCR of CSB at PN19 and PN20 in IMR-90 fibroblasts knocked down for CSB (shCSB#1 and shCSB#2) and scramble control (shSCR). n = 3 independent experiments, mean ± SD, values are reported in the Source Data files; two-way ANOVA (F = 28.24, DFn = 2, DFd = 12, p < 0.0001) with post-hoc Tukey’s test vs. shSCR. d Direct correlation between p21Waf1 and HTRA3 transcript in control shSCR (from data in Supplementary Fig. 5b, c). e Cumulative population doubling of serially passaged IMR-90 (starting at PN18, n = 3 independent cultures). f Quantification of SA-ß-gal+ cells from PN19 to PN28 (cultures stopped growing at PN26 with shCSB#1 and at PN24 with shCSB#2); n = 1160-4480 cells/condition from three independent experiments, mean ± SEM; two-way ANOVA (PN19-24: F = 542.3, DFn = 2, DFd = 40677, p < 0.0001. PN25-26: F = 78.71, DFn = 1, DFd = 5114, p < 0.0001) with post-hoc Tukey’s (PN19-24) or Sidak’s (PN25-26) tests vs. the respective shSCR; n.a = not applicable. Arrows indicate the initial burst of SA-ß-gal+ staining upon CSB silencing. SA-ß-gal+ cells in shSCR at PN19-PN20 probably result from response to lentiviral infection68. RT-qPCR of g p21Waf1 and h HTRA3 at PN19 and PN20. n = 3 independent experiments, mean ± SD; two-way ANOVA (p21Waf1: F = 44.26, DFn = 2, DFd = 12, p < 0.0001. HTRA3: F = 35.04, DFn = 2, DFd = 12, p < 0.0001) with post-hoc Tukey’s test vs. shSCR. Data in panels c, g, h (which are limited to PN19 and PN20) are extracted from panels in Supplementary Fig. 5a–c. i WB of HTRA3, p21, HTRA2, POLG1, p16, and CSA at PN20. Samples on the same blot are framed; each frame displays the respective GAPDH or ß-tubulin used as a loading control (middle blot, GAPDH F-C staining). High levels of p16 in shSCR are compatible with lentiviral infection69. j Linear regression of each individual value in Supplementary Fig. 5a (CSB, x-axis) vs. log10-transformed values in Supplementary Fig. 5b (p21Waf1, y-axis). Source data are provided as Source Data files.
Fig 3: CSB-dependent senescence is specific to the DDR/p21 pathway. a Enumeration of endogenous ?-H2AX and 53BP1 foci/cell at PNs preceding or during CSB depletion. n = 50–70 cells from three independent experiments; mean ± SEM; one-way ANOVA (?-H2AX: F = 3.209, DFn = 3, DFd = 220, p = 0.0239; 53BP1: F = 5.603, DFn = 3, DFd = 220, p = 0.001) with post-hoc Tukey’s test vs. the respective PN16. b Representative confocal acquisitions of irradiated (10 Gy) and non-irradiated (non-IR) IMR-90 immunostained for ?-H2AX (green) and 53BP1 (red), counterstained with Hoechst (blue, nuclei), after maximum intensity projection with the Imaris software; scale bar = 20 µM. Percentage of cells with 0, 1, 2, 3, or >3 (c) ?-H2AX or d 53BP1 foci per nucleus. n = 90 cells from three independent experiments. Mean ± SEM; two-way ANOVA (?-H2AX: F = 35.77, DFn = 4, DFd = 20, p < 0.001; 53BP1: F = 57.63, DFn = 4, DFd = 20, p < 0.0001) with post-hoc Sidak’s test vs. the respective non-IR. e Quantification of SA-ß-gal+ cells of irradiated and non-IR IMR-90 at PN17. n = 180–220 cells/condition from three independent experiments, mean ± SEM; unpaired Student’s t-test (two-tailed) (t = 36.28, DF = 408), p-value vs. non-IR (f) Immunoblots of CSB, HTRA3, p21, and POLG1, HTRA2, p16 (GAPDH F-C staining, loading control, under each blot) in irradiated and non-IR fibroblasts. g Scheme indicating the palbociclib target in the p16 pathway to senescence. h Quantification and i representative images of SA-ß-gal+ staining of palbociclib-treated and untreated IMR-90 cells, scale bar = 200 µM. Early-passage fibroblasts (PN20) in normal medium (CTL) or in the presence of 0.2, 0.5, 1, or 5 µM palbociclib for 7 days followed by 1 day of drug withdrawal; n = 800 cells (CTL) and n = 320–380 cells (palbociclib-treated samples) from three independent experiments, mean ± SEM; one-way ANOVA (F = 195.1, DFn = 4, DFd = 2236, p < 0.0001) with post-hoc Tukey’s test. RT-qPCR of j HTRA3, k CSB, and l p21Waf1 and p16Ink4. n = 3 independent experiments, mean ± SD; RM one-way ANOVA (HTRA3: F = 53.07, DF = 4, p < 0.0001; CSB: F = 29.47, DF = 4, p < 0.0001; p21Waf1: F = 13.36, DF = 4, p = 0.0015; p16Ink4: F = 1.587, DF = 4, p = 0.2531) with post-hoc Tukey’s test vs. CTL. m WB of HTRA3, HTRA2, p21, p16, and CSB. Samples on the same blot are framed; each frame displays the respective GAPDH (F-C staining) used as a loading control. Source data are provided as Source Data files.
Fig 4: Overexpression of HTRA3 and mitochondrial impairment in replicative senescence. a Cumulative population doubling of IMR-90 fibroblasts (starting from PN15). Senescence corresponds to plateau (proliferative arrest). Cells analyzed at PNs identified with black arrows; n = 3 independent cultures; mean ± SD, values are reported in the Source Data file. b Percent of SA-ß-gal+ cells; n = 1100–1340 cells (PN16-PN27) and n = 180–410 cells (PN31-PN35) from three independent experiments, mean ± SEM (values indicated on the top of columns); one-way ANOVA (F = 768.9, DFn = 5, DFd = 5551, p < 0.0001) with post-hoc Tukey’s test vs. PN16. c Quantification of mean HTRA3 fluorescence intensity per cell at indicated PN (representative images in Supplementary Fig. 1d). d Sublocalisation of HTRA3 and mitochondria in early-passage (PN16) and senescent (PN35) IMR-90 fibroblasts with SIM (one plan of a z-stack acquisition for each cell). Cells immunolabelled for ATP synthase ß (green) to reveal mitochondria, and HTRA3 (red); nuclei counterstained with Hoechst (blue). Scale bars = 10 µm. For each cell, a x2.5 magnification of a region (1 or 2) is shown on the right with immunostaining for ATP synthase ß, HTRA3, and merge (representative arrowhead for HTRA3/ATP synthase ß colocalization, arrow for extra-mitochondrial HTRA3, and triangle for mitochondria with no HTRA3 signal detection). e Immunoblot of HTRA3, and GAPDH (loading control, reprobing after stripping). a-HTRA3 antibody recognizes long and short isoforms (Supplementary Fig 1e). f Quantitative RT-qPCR of HTRA3 (short and long form), p21Waf1, p16Ink4, and IL-6 transcripts. n = 3 independent experiments; mean ± SD; one-way ANOVA (HTRA3s: F = 13.12, DFn = 5, DFd = 12, p = 0.0002; HTRA3l: F = 29.12, DFn = 5, DFd = 12, p < 0.0001; p21Waf1: F = 63.24, DFn = 5, DFd = 12, p < 0.0001; p16Ink4: F = 38.71, DFn = 5, DFd = 12, p < 0.0001; IL-6: F = 29.75, DFn = 5, DFd = 12, p < 0.0001) with post-hoc Tukey’s test vs. PN16 (p-values on the top of scatter plots/columns). Quantification of mean fluorescence intensity (mFI) of g HTRA2 and h POLG1 immunolabeling per cell (representative images in Supplementary Fig. 3c, d). i WB of POLG1, HTRA2, each with the loading control GAPDH. IFs: n = 30–50 cells from three independent experiments; mean ± SEM; one-way ANOVA (c F = 58.32, DFn = 5, DFd = 194, p < 0.0001; g F = 30.75, DFn = 5, DFd = 194, p < 0.0001; h F = 16,67, DFn = 5, DFd = 193, p < 0.0001) with post-hoc Tuckey's test. IF measurements include normalization to cell size. Source data are provided as Source Data files.
Supplier Page from MilliporeSigma for Anti-HTRA3 antibody produced in rabbit