Fig 2: The effect of knockdown of DLAT and CDKN2A on cell proliferation, migration and invasion. (A) Western blot indicated that DLAT protein expression levels were decreased after knockdown of DLAT in HepG2 and SMMC7721 cells (above). Bands were quantified with Image J software and evident from the quantification of Histogram. β-actin was used as a loading control. Data are presented as mean ± SD, n=3. ***P< 0.001 (down). (B) Western blot indicated that CDKN2A protein expression levels were decreased after knockdown of CDKN2A in HepG2 and SMMC7721 cells. (above). Bands were quantified with Image J software and evident from the quantification of Histogram. β-actin was used as a loading control. Data are presented as mean ± SD, n=3. ***P< 0.001 (down). (C, D) CCK-8 assay showed that cells proliferation was inhibited. Colony formation assays, with representative images (E) and bar graphs (F) from three independent experiments, demonstrated that cells proliferation was inhibited in cells with DLAT and CDKN2A knockdown. Data expressed as mean ± SD. ***P< 0.001. (G-J) Comparison of the migration and invasion of HepG2 and SMMC7721 cells using transwell compartments. Representative images (G, I) and statistics (H, J) of migration and invasion assay were shown.

Fig 3: (A) The right panels: cells were transplanted with sh‐NC, sh‐LINC01116, pcDNA3.1‐ miR‐9‐5p or CCNE1 plasmids (n = 3). The left panels: the weight and volume of each tumour were measured (*p < 0.05, **p < 0.01, ***p < 0.001). (B) The tumour weight in the sh‐LINC01116 group was decreased, and the pcDNA3.1‐miR‐9‐5p/CCNE1 group was increased (*p < 0.05, **p < 0.01, ***p < 0.001). (C) The tumour volume was decreased in the sh‐LINC01116 group but increased in the pcDNA3.1‐miR‐9‐5p/CCNE1 group (*p < 0.05, **p < 0.01, ***p < 0.001). (D and E) The relative expression level of LINC01116 and miR‐9‐5p in the tumour tissue of BALB/C nude mice transplanted with sh‐NC, sh‐LINC01116, pcDNA3.1‐miR‐9‐5p or CCNE1 plasmids was achieved by qRT‐PCR (*p < 0.05, ***p < 0.001). (F) Protein expression of CCNE1, CDK2, Ki‐67 MCM7, PCNA and p16 in the tumour tissue of BALB/C nude mice, as measured by the Western blot analysis.

Fig 4: HDAC3 Overexpression delays chondrocyte aging and OA progression. a, b Western Blotting analysis of HDAC3, p16INK4a, p21, and p53 expression in the primary chondrocytes of mice which transfected adenovirus containing HDAC3 (Ad-HDAC3) or Ad-NC cultivated on 10:1 PDMS substrates (n = 5). c Representative images of safranin O/fast green and immunofluorescence staining of HDAC3 in cartilage of mice intra-articularly injected with AAV-NC or AAV-HDAC3 after DMM surgery. Scale bars: 50 µm. d Quantification of HDAC3-positive chondrocytes based on staining results in (c) (n = 5). e Quantification of the OARSI scale based on staining results in (c) (n = 5). f Representative images of immunofluorescence staining of p16INK4a, p21, and p53 in cartilage of mice intra-articularly injected with AAV-NC or AAV-HDAC3 after DMM surgery. Scale bars: 50 µm. g Quantification of p16INK4a, p21, and p53-positive chondrocytes based on staining results in (f) (n = 5). h Representative images of immunofluorescence staining of PINK1, Parkin, and Lc3 in cartilage of mice intra-articularly injected with AAV-NC or AAV-HDAC3 after DMM surgery. Scale bars: 50 µm. i Quantification of PINK1, Parkin and Lc3-positive chondrocytes based on staining results in (h) (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001. OA osteoarthritis, DAPI 4’,6-diamidino-2-phenylindole, OARSI Osteoarthritis Research Society International, AAV-HDAC3 adenovirus expressing small hairpin HDAC3, AAV-NC negative control

Fig 5: Effects of ARG2 on senescence of skin fibroblast cells. (A) Effect of ARG2 knockdown on the expressions of cellular senescence markers, p16 and p21, in skin fibroblasts. Cells were transfected with an ARG2 siRNA and then treated with H2O2 (200 μM) for 24 h. (B) Effect of ARG2 overexpression on the expressions of the cellular senescence markers, p16 and p21, in skin fibroblasts. Cells were transfected with a plasmid containing a flag-tagged ARG2. (C) Representative images of H2O2-treated skin fibroblasts stained for senescence-associated β-galactosidase after being transfected with an ARG2 siRNA (left) or an ARG2 overexpression plasmid (right). # represents the number of senescent cells. (D) Effect of restoring ARG2 levels on senescence of skin fibroblasts. Cells were transfected with an ARG2 siRNA, treated with H2O2 for 24 h, and then transfected with a flag-tagged ARG2 plasmid. Protein levels were determined using Western blotting. A * p value < 0.05 and ** p value < 0.01 were considered statistically significant and error bars represent ± SD.