Mcp 1

For immunohistochemical analysis, the dewaxed sections were boiled in 10 mmol/L citrate buffer for 20 min for epitope retrieval following washing three times in PBS. After incubating in 3% hydrogen peroxide, the sections were blocked with 1% BSA for 1 hour. The slices were incubated with anti-rat antibodies against IL-10 (1:200, Servicebio, China), TGF-β3 (1:200, Servicebio, China), IL-6 (1:400, Servicebio, China), TNF-α (1:300, Servicebio, China), IFN-γ (1:150, Servicebio, China), α-SMA (1:1000, Servicebio, China), MCP-1 (1:500, Servicebio, China), CCR2 (1:800, Servicebio, China) and CCRL2 (1:200, Servicebio, China) at 4°C overnight and then incubated with HRP labeled Goat anti-rabbit IgG general secondary antibody (DAKO, Denmark). Finally, the antibody binding was visualized by using a DAB kit (Thermo Scientific). All sections were imaged using an automatic slide scanning system (AxioScan.Z1, Zeiss, Germany) at 20X magnification.

Antibodies to Kim-1, p21 and H3K36me3 were purchased from Abcam Inc. (Cambridge, United Kingdom). Antibodies to cleaved Caspase 3, Cyclin D1, SMYD2, p-p53, p53, p-STAT3, STAT3, p-NFκBp65, and Tubulin were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to PCNA, F4/80, MCP-1, ICAM-1 and TUNEL were purchased from Servicebio (Wuhan, China). Antibodies to NGAL was purchased from R and D SYSTEMS (Minneapolis,United States). Antibodies to GAPDH were purchased from Arigo Biolaboratories Corp. (Shanghai, China). Antibodies to BAX was purchased from Absin Bioscience Inc. (Shanghai, China). Antibodies to Histone-H3 was purchased from Merck (Darmstadt, Germany). Antibodies to NFκBp65 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to BCL-2 was purchased from Proteintech Group Inc. (Chicargo, United States). SMYD2 siRNA and scramble siRNA were purchased from GenePharma Co.Ltd. (Shanghai, China). AZ505 was purchased from MedChemExpress (New Jersey, United States). Cisplatin and all other chemicals were purchased from Sigma (St. Louis, MO).

Renal tissues and cells were lysed using RIPA lysate (Servicebio). The BCA method was used to quantify total protein. Proteins were separated on polyacrylamide gels , then moved to a polyvinylidene fluoride membrane 5% skim milk was used to prevent non-specific binding to the membrane for 2 h at room temperature, followed by incubation overnight with one of the following primary antibodies: EZH2 (5246S; 98 kDa; 1:1,000; CST), H3K27me3 (9733S; 17 kDa; 1:1,000; CST), H3(17168-1-AP; 17kDa; 1:2000; Proteintech), cleaved-caspase3(19677-1-AP; 17 kDa; 1:1,000; Proteintech), Bcl2 (12789-1-AP; 26 kDa; 1:1,000; Proteintech), Bax (GB114122; 21 kDa; 1:800; Servicebio), MCP-1(GB113239; 11 kDa; 266 1:500; Servicebio), Sox9 (67439-1-Ig; 56 kDa; 1:1000; Proteintech), β-catenin (GB12015; 92 kDa; 1:500; Servicebio), surviving (GB11177; 16 kDa; 1:1,000; Servicebio), GAPDH (GB12002; 37 kDa; 1:1,000; Servicebio) at 4 °C. Horseradish peroxidase-coupled secondary antibodies were incubated for 1 h with the protein bands after being washed by TBST, followed by visualization using an ECL kit (Biosharp, China) on a ChemiDoc MP system (Bio-Rad, Foster City, CA, USA). The relative density of proteins was measured using ImageJ software (1.8.0). All experiments were performed using three replicates.