Fig 2: Near total deletion of CCN2 resulted in reduced DNA damage and p53 activation 4 h after IRI. (A,B) Representative micrographs of mouse renal cortex stained with gamma H2AX (?H2AX; (A) and phosphorylated p53 (p-p53; B). Quantification of ?H2AX (A) and p-p53 (B) staining showed that increased DNA damage and p53 activation in IRI kidneys decreased in CCN2 KO mice compared with WT mice. Additionally, p-p53 expression correlated with CCN2 mRNA in CCN2 KO kidneys (B). (C,D) Quantitative real-time polymerase chain reaction (qPCR) analysis showed that mRNA expression levels of p21CIP1 (p21) and GADD45A increased by IRI in WT and in CCN2 KO kidneys. Data are expressed as the mean ± SEM (n= 6 for WT sham; n = 6 for WT IRI; n = 4–5 for KO sham; n = 6–7 for KO IRI). TBP was used as an internal control. * p < 0.05, ** p < 0.01, and, *** p < 0.005. Bar = 100 µm.

Fig 3: Silencing of CCN2 alleviated oxidative stress response induced by AR-injury in cultured PTECs. (A) Representative Western blotting of protein extracts from cultured cells for 4-HNE, nitrotyrosine, and p-p53 showed that increased oxidative stress response and DDR in AR-injured PTECs was alleviated by silencing of CCN2. Data represent three independent experiments. (B) Malondialdehyde (MDA) levels indicated that increased oxidative stress response in AR-injured PTECs was alleviated by silencing of CCN2. Data represent six independent experiments. Data are expressed as the mean ± SEM. *** p < 0.001.

Fig 4: The increase of HDAC9 was associated with bone and fat imbalance in bone aging. a Micro-CT analyses of bone mass of trabecular and cortical bone thickness in the femora of 2-month-old (young) and 16-month-old (aged) mice. Bone mineral density (BMD), trabecular bone volume (BV/TV), and cortical bone thickness (Ct.Th) were performed. Scale bar = 1 mm. b–d Immunofluorescent staining of OCN (b), PPAR-? (c), and TRAP (d) was performed in the bone marrow from young and aged mice, and the positive signals were quantitatively analyzed. Scale bar = 50 µm. e Expressions of the senescence-related proteins, p53 and p-p53, in the bone marrow from young and aged mice were examined by western blotting. f Expressions of the senescence-related proteins, p53 and p-p53, in BMMSCs from young and aged mice were examined by western blotting. g Alizarin Red staining was performed, and quantification of mineralized nodules was analyzed in young and aged BMMSCs. h Oil Red O staining was performed, and quantification of lipid droplet positive ratio areas was analyzed in young and aged BMMSCs. Scale bars = 100 µm. i, j Expression of HDAC9 (i) and acetylation sites of H3K, including H3K9, H3K18, and H4K16 (j), were examined by western blotting. The data are presented as the means ± SD of each independent experiment performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t test

Fig 5: Mechanisms involved in IRI linked with CCN2. Inhibition of CCN2 reduced the expression of ?H2AX and p-p53 marking reduced DNA damage and DDR in the immediate early response following IRI. In the early phase upon IRI, this was followed by sustained reduction in DNA damage and DDR, along with reduced anti-apoptosis. This was associated with reduced oxidative stress response. Dotted arrows are used to acknowledge that the exact mechanisms by which CCN2 contributes to DNA damage and DDR remain to be established.