Fig 2: RG-7112 treatment of IVD pellet cultures.(A) Representative images indicating how we deemed positive and negative p16INK4a (a), Ki-67 (d), and caspase-3 (g) staining. (b, e and h) Magnified images of (a, d and g). Arrow heads indicate positive (green) and negative (red) staining. (c, f and i) no secondary antibody control photomicrographs. Quantification of (B) p16INK4a (n = 7), (C) Ki-67, and (D) caspase-3 expression, (n = 8). Scale bars: 20 μm in (A). The cells were from degenerating IVDs as indicated in Table 2. Values are presented as mean ± SEM in (B–D). * Indicates a significant difference assessed by the two-tailed Student's t-test: p<0.05; **p<0.01 and ****p<0.0001.Figure 1—source data 1.Excel file containing the percentage of p16Ink4a, ki-67 and caspase three positive in NP and AF cells control and treated with RG-7112.

Fig 3: The EML4–ALK21S mutant exhibits impaired capability in tumor formation.a Schematic illustration of tumor formation assay in nude mice. See “Materials and methods” for details. b Photos of subcutaneous tumors derived from control, GFP–EML4–ALK and GFP–EML4–ALK21S groups. Scale bar, 1 cm. n = 7 for each group. c Growth curves of the subcutaneous tumors. d Statistical analysis of tumor weights. e Representative photos for ALK immunostaining in subcutaneous tumors derived from control, GFP–EML4–ALK and GFP–EML4–ALK21S groups. Scale bar, 50 μm. f Representative fluorescence photos for subcutaneous tumors derived from GFP–EML4–ALK and GFP–EML4–ALK21S groups. The GFP–EML4–ALK or GFP–EML4–ALK21S was visualized by confocal microscopy. Nucleus was stained with DAPI (blue). Scale bar, 10 μm. g Representative images of low, medium, high expression of Ki-67. Scale bar, 50 μm. h Representative photos for Ki-67 immunostaining in subcutaneous tumors derived from control, EML4–ALK and EML4–ALK21S groups. Scale bar, 50 μm. i Statistical analysis of Ki-67 staining. j Representative images of low, medium, high expression of p-STAT3. Scale bar, 50 μm. k Representative photos for p-STAT3 immunostaining in subcutaneous tumors derived from control, GFP–EML4–ALK and GFP–EML4–ALK21S groups. Scale bar, 50 μm. l Statistical analysis of p-STAT3 immunostaining. All data were shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Ctrl, control; WT, GFP–EML4–ALK; 21S, GFP–EML4–ALK21S.

Fig 4: RUNX1 characterization of human PVR membranes. (a) Funduscopic image of a patient with PVR. Large preretinal membranes are observed creating retinal folds (left). Representative macroscopic appearance of human specimen obtained during PVR surgery (right). Note high degree of pigmentation denoting the presence of RPE cells within the pathological excised tissue. Scale bar: 400 μm. (b) RUNX1 positive cells are clearly identified in human PVR specimens obtained from four different donors. Positive Ki67 labelling was found in those cells with an active proliferative state within the tissue. Scale bar: 400 μm. (c) Cell proliferation and sprouting in human PVR explants were evaluated. A reduction in sprouting distance from the specimen is observed 7 days in explants treated with 150 μM Ro5-3335 (**p < 0.001, two-way ANOVA; n = 3 represented as mean ± SEM). Scale bar: 400 μm. (d) C-PVR cells from three different donors showed positive staining with RUNX1 antibody. Scale bar: 400 μm. (e) RUNX1 inhibition with Ro5-3335 inhibitor reduces C-PVR cells proliferation in vitro in a dose-dependent manner (25 μM, 50 μM and 100 μM), at 48 and 72 h (**p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA; n = 4 represented as mean ± SEM).

Fig 5: Ezh2 represses cell senescence inducer genes in MSPCs via H3K27me3. a CD45−GFP+ and CD45−GFP− mesenchymal stem/progenitor cells (MSPCs) were separately isolated from femoral metaphyses from 4-week-old male Nestin-GFP mice, and the total RNA was subjected for PCR array analysis of the chromatin modification enzymes. b Validation of messenger RNA changes by qRT-PCR analysis. c–g Chromatin immunoprecipitation (ChIP)-qPCR assays with H3K27me3 antibody or IgG antibody were performed using CD45−GFP+ and CD45−GFP− MSPCs. Schematic representation of the INK4a-ARF-INK4b locus in mouse c. Rectangles indicate coding exons of the three genes (p15 INK4b , p19 ARF and p16 INK4a) separated by intronic sequences (black horizontal line). The regions chosen for PCR amplification by the primers in ChIP-qPCR assays are also indicated. ChIP and input DNA were measured using RT-qPCR with specific primers targeting the promoter regions of senescence-associated genes (d–f). ChIP and input DNA were measured using quantitative RT-PCR with specific primers targeting the promoter regions of osteogenic genes (g). h–m Quantitative RT-PCR analysis of p16 INK4a (h), p21 CIP1 (i), ki67 (j), p53 (k), Runx2 (l), Osteocalcin (m) expression in the sorted CD45−GFP+ and CD45−GFP− MSPCs. n = 3, Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests