Fig 2: Silencing RNF138 expression promotes H2O2-induced injury and silencing of miR-29b-3p ameliorates it by upregulating RNF138. (A) The silencing efficiency of RNF138 in HTM cells was verified by reverse transcription quantitative polymerase chain reaction and western blotting. *P<0.05 vs. si-NC group. (B) HTM cell viability in each group was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. (C) The apoptotic rate of HTM cells in each group was evaluated by flow cytometry analysis. (D) The levels of apoptosis-related proteins in HTM cells of each group were determined by western blotting. (E) Reactive oxygen species generation in HTM cells of each group was detected via 2,7-dichlorodihydrofluorescein diacetate staining. (F) The protein levels of extracellular matrix-related genes in HTM cells of each group were evaluated by western blot analysis. *P<0.05 vs. control group; #P<0.05 vs. H2O2 and H2O2 + NC groups; &P<0.05 vs. H2O2 + miR-29b-3p inhibitor group. RNF138, E3 ubiquitin-protein ligase RNF138; miR, microRNA; HTM, human trabecular meshwork; si-, small interfering RNA; NC, negative control; COL1A1, collagen a-1(I) chain; COL1A2, collagen a-2(I) chain.

Fig 3: Effect of poly- and oligosaccharide fractions from Ulva sp. on (a) COL1A1, (b) COL1A2, (c) MMP-1, (d) COL3A1, and (e) TIMP-1 mRNA expression levels in fibroblasts. NHDF (n = 4) were cultured for 48 h with the fractions at two concentrations (50 and 1000 µg/mL) for the assessment of genes. Significant differences between fractions and control are indicated by * p < 0.05 and ** p < 0.01.

Fig 4: Impaired synthesis of extracellular matrix (ECM) components and disrupted chondrogenic differentiation of TRIP11-mutant cells.(A) Number of chondrogenic nodules at day 7 of transdifferentiation of fibroblasts (fibroblast-induced chondrocytes, FDC) in controls and patients. (B) Size of the chondrogenic nodules at day 7, as exemplified for case 6 by inverse microscopy of live cells in (C). Scale bar: 100 µm. (D) Alcian blue staining of transdifferentiated patient-derived primary and matched control FDC cultures at days 1–7 of chondrogenic differentiation demonstrates reduced matrix production in patient cells. Scale bars: 150 µm. (E) Quantification of total glycosaminoglycan/proteoglycan content in FDCs; data points represent the mean of triplicate values (n = 3); error bars indicate ± SD. (F and G) Quantitative ELISA shows that secreted markers of terminal differentiation, COL10A1 and pro-MMP13, are significantly reduced in supernatants of patient-derived cultures at late time points of the transdifferentiation protocol. CTL, controls 1 and 2; ODCD, odontochondrodysplasia cases 3, 6, and 10; ACG1A, achondrogenesis 1A cases A1 and A2. Data points represent the mean of quadruplicate values (n = 4); error bars indicate ± SD. (H) Quantitative ELISA of pro-collagen, type 1, a-1 (pro-COL1A1) in conditioned FDC supernatants. Data represent mean ± SD of quadruplicates (2-way ANOVA with Bonferroni’s post hoc test); *P = 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Bromodomain inhibitors reduce radiation-induced DGKA and pro-fibrotic marker expression in BJ cells. (A) Nuclear H3K27ac expression in BJ cells treated with bromodomain inhibitors. (B) Quantitative ACT-seq signals for H3K27ac enrichment at the DGKA locus in BJ cells. (C–E) Relative mRNA expression of DGKA (C), COL1A1 (D), and COL3A1 (E) in BJ cells. For (A–E), cells were pre-treated with DMSO, CBP (10 µM), or JQ1 (5 µM) for 48 h, irradiated with 6 Gy, and analyzed after an additional 48 h with concurrent drug treatment. (F) Secreted COL1A1 was measured by ELISA. Cells were pre-treated with DMSO, CBP (10 µM), or JQ1 (5 µM) for 48 h, irradiated with 6 Gy, and the conditioned medium was harvested after a further 72 h with concurrent drug treatment. (G,H) Relative mRNA expression of CBP and p300 (G) as well as DGKA, COL1A1, and COL3A1 (H) in BJ cells after silencing CBP and p300. Cells were pre-treated with siRNAs directed against both enzymes for 48 h, irradiated with 6 Gy, and harvested after an additional 48 h. Statistical data are presented as mean ± SEM from three (A,B,G,H), six (C–E) or four (F) biological replicates. Statistical significance (* p < 0.05, ** p < 0.01, and *** p < 0.001) was determined by one-tailed Student’s t-test.