Western Blot Analysis

Kidney and cell culture samples were lysed with RIPA buffer containing protease inhibitors cocktail and centrifuged at 12000 g for 5 min. Then supernatants were collected. Protein concentration estimations were determined using the BCA protein assay kit (Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat dry milk and incubated with specific primary antibodies for α-SMA (ab5694, Abcam, United Kingdom), FN (ab45688, Abcam, United Kingdom), Collagen I (Col I, ab21286, Abcam, United Kingdom), NF-κB p65 (#8242, Cell Signaling Technology, United States), Phospho-NF-κB p65 (Ser536, ab86299, Abcam, United Kingdom), smad3 (ab40854, Abcam, United Kingdom), Phospho-smad3(ab52903, Abcam, United Kingdom), CyclinD1 (ab134175, Abcam, United Kingdom), PCNA (ab18197, Abcam, United Kingdom) and p21 Waf1/Cip1 (ab109199, Abcam, United Kingdom), and Phospho-histone H3 (p-H3, #9701, Cell Signaling Technology, United States), E-cadherin (#3195, Cell Signaling Technology, United States) Vimentin (ab92547, Abcam, United Kingdom), GAPDH (#5174, Cell Signaling Technology, MA, United States), Snail1 (#271977, Santa Cruz, CA, United States) and Twist (ab49254, Abcam, United Kingdom) followed by incubation with the secondary antibody (horseradish peroxidase-labeled IgG anti-rabbit/mouse antibody, Cell Signaling Technology). Bands were visualized by enhanced chemiluminescent substrates (ECL, Thermo Scientific) and analyzed using Image J software (National Institutes of Health, Bethesda, MD, United States).

Data were presented as means ± SEM values. t-Test and One-Way ANOVA followed by Student–Newman–Keuls post hoc test were used for comparisons between groups. All statistical calculations were made using Graphpad Prism (7.0, La Jolla, CA, United States). P-value < 0.05 was accepted as statistically significant.