P erk

Total protein was extracted from RAW264.7 macrophages and primary peritoneal macrophages using radio immunoprecipitation lysis buffer (RIPA, Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors (Solarbio, Beijing, China). The protein was collected, and its concentration was measured using a Pierce BCA protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gels and subsequently transferred onto PVDF membranes. Blocking was with TBST containing non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies against NRF2, p-AKT, AKT, iNOS (Cell Signaling Technology, Danvers, MA, USA), and HO-1, NQO-1, COX-2, p-GSK-3β, GSK-3β, p-AMPK-α1, AMPK-α1, p-ERK, EKR, p-JNK, JNK, p-P38, P38, p-NF-κB p65, NF-κB p65, and GAPDH (ABclonal, Wuhan, China) were diluted to a ratio of 1:2000. Before adding secondary antibodies (goat anti-rabbit or goat anti-mouse) diluted to a concentration of 1:10,000 (ABclonal, Wuhan, China), the membranes were washed four times with TBST for 15 min each time. Membranes were visualized using an Amersham Imager 600 (a gel imaging system from GE Co., Fairfield, CT, USA) after applying enhanced chemiluminescence. For detailed information of WB procedures, refer to our previous study [50 (link)].

KUP5 and AML12 cells used for protein extraction were cultured in 6-well plates and treated with CDs concentrations of 0, 100 and 400 μg/mL. The upper medium was discarded and the cells were lysed as described previously [44 (link)]. The protein content was analyzed using the bicinchoninic acid (BCA) method and equal amounts (30 μg) of protein sample were loaded onto a 12.5% SDS-PAGE gel for separation, transferred to a PVDF membrane, and sealed with 5% skim milk powder diluted with TBST for 1 h. The samples were then incubated with primary antibody (1:1000) for Cleaved-caspase3, p62, Beclin1 (Cell Signal Technology (CST), USA), Bax, Bcl-2, GAPDH, TFEB, H3, p-mTOR, or p-ERK (ABclonal, Wuhan, China) at 4 °C overnight. Thermo32106ECL luminescence (Thermo Fisher Scientific, USA) measurements were performed after co-incubation with the corresponding secondary antibody (V: V = 1:10,000) at room temperature for 1 h. The Tanon MP imaging System (Tanon 5200, Shanghai, China) and ImageJ 1.53a were used for image acquisition and strip gray scale quantification.

VPA was obtained from MedChemExpress (New Jersey, USA). GAPDH, JNK, p-JNK, p38, p-p38, ERK, p-ERK, SIRT3, CD3, LY6G and CX3CR1 antibodies were purchased from ABclonal (Wuhan, China), and antibodies against CX3CL1 and Lamp-2b were bought from Abcam. Osteocalcin antibody was purchased from Takara (Japan, M173). Penicillin-streptomycin was purchased from Solarbio (Beijing, China). The RANKL and M-CSF were bought from R&D Systems (Minnesota, USA). The cell culture plates were from NEST (Jiangsu, China). Minimum Essential Medium Alpha (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). CCK-8 (Kumamoto, Japan) was bought from Dojindo. The enzyme-linked immunosorbent assay (ELISA) kits were purchased from CUSABIO and Elabscience (Wuhan, China). TRAP staining kit was obtained from Sigma–Aldrich (St. Louis, MO, USA).

Tissues and cells were lysed, and protein concentrations were determined. Antibodies: HRD1, c-caspase12, Flag, Myc, HA, PI3k, Akt and p-Akt (Proteintech); β-actin (Bimake); p-PI3k (Bioworld); TMEM2 (AVIVA); p-PERK, PERK, c-PARP, c-caspase3, ATF6, CHOP, p-IRE1, and IRE1 (ABclonal); and ubiquitin (Cell Signaling Technology). Gel-Pro Analyzer version 4.0 (Media Cybernetics, MD, USA) was used for protein quantification.

Proteins were extracted from cells and tissues using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Solarbio) and determined using the Bradford protein assay kit (Beyotime). Cell lysates containing equal amount of proteins (30 µg) were separated via SDS/PAGE gels and transferred to 0.22 µm PVDF membranes (Merck Millipore Ltd). Blots were blocked with 5% milk for 30min at 37 °C, and incubated with primary antibodies, including E-cadherin (CST. No, 3195S, 1:1000), Vimentin (CST. No, 5741S, 1:1000), snail (CST, no, 3879S, 1:1000), MMP2 (CST. No, 40994S, 1:1000), MMP9 (CST. no, 13667S, 1:1000), NKCC1 (Proteintech, no,13884-1-AP 1:10000), JNK (Santa Cruz, no, 7345, 1:200), p-JNK (Santa Cruz, no, 6254, 1:200), ERK (ABclonal, no, A19630, 1:1000), p-ERK (ABclonal, no, APO485, 1:1000), P38 (ABclonal, no, A4771, 1:1000), p-P38 (ABclonal, no,AP05261:1000), and GAPDH (Cell Signaling Technology, 1:10000). The blots were probed with secondary antibody (Cell Signaling Technology 1:5000 dilution) for 30min at 37 °C. The positive bands were detected by ECL kit (Pierce).

Dulbecco’s modified eagle medium (DMEM) and penicillin–streptomycin were supplied from Gibco (Grand Island, NY, USA), while fetal bovine serum (FBS) was purchased from Thermo Scientific Company (Waltham, MA, USA). Apoptosis annexin V and PI was obtained from Biolegand (San Diego, CA, USA.), whereas 3MA, MDC, DCF-DA, and cisplatin were obtained from Sigma-Aldrich (St. Louis, MO, USA). DCF and NAC were obtained from MedChemexpress (Princeton, NJ, USA). Aldose reductase inhibitor screening kit was purchased from Biovision (Waltham, MA, USA). KATO III cell lines was obtained from American Type Culture Collection (Manassas, VA, USA). Antibodies specific to AKR1C1, AKR1C3, PARP1, LC3B-II, Nrf2, HO-1, SOD1, p38, p-p38, ERK, p-ERK, NFκB, STAT3, c-Jun, Akt, p-Akt, cyclin D1, cIAP2, Bcl-2, Bcl-xL were purchased from Abclonal (Woburn, MA, USA).