Stat3

Western blot analysis was performed as previously described 26 (link). The same amount of protein was separated by SDS gel and transferred to PVDF membranes. The membranes were blocked at room temperature in goat serum for 1 hour. And then each membrane was incubated with specific primary antibodies against ALDH3A1, E-cadherin and vimentin (Abcam, USA), p-STAT3 (Tyr705), MMP3 (Cell Signaling Technology, USA), IL-6, snail, STAT3 and GAPDH (ABclonal, USA) overnight. Next, the membranes were incubated with secondary antibodies (ABclonal, USA) and visualized using enhanced chemiluminescence reagents (Bio-Rad, USA).

Antibodies against PD-L1 (A11273, dilution for western blot, 1:1000; dilution for IHC analysis, 1:100; dilution for IF analysis, 1:100) and STAT3 (A11867, dilution for western blot, 1:1000; dilution for IF analysis, 1:100) were obtained from ABclonal (Wuhan, China). Antibodies against CD163 (bs-2527R, dilution for IHC analysis, 1:100), CD206 (bs-4727R, dilution for IHC analysis, 1:100), iNOS (bs-2072R, dilution for IHC analysis, 1:100) and IFNγ (bs-0480R, dilution for IHC analysis, 1:100) were obtained from Bioss (Beijing, China). Antibodies against Phospho-STAT3 (Tyr705) (9145S, dilution for western blot, 1:1000; dilution for IHC analysis, 1:100; dilution for IF analysis, 1:100; dilution for ChIP, 1:100) were obtained from Cell Signaling Technology (USA). Antibodies against β-actin (TA-09, dilution for western blot, 1:2000), peroxidase-conjugated goat anti-rabbit IgG (ZB-2301, dilution for western blot, 1:5000) and Peroxidase-conjugated goat anti-mouse IgG (ZB-2305, dilution for western blot, 1:5000) were obtained from ZSGB‐BIO (Beijing, China). Alexa Fluor 594 donkey anti-rabbit IgG (A21207, dilution for IF analysis, 1:1000) and Alexa Fluor 488 donkey anti-mouse IgG (A21202, dilution for IF analysis, 1:1000) were obtained from Invitrogen (USA).

Cells were seeded into 60 mm cell culture plates at a density of 4 × 10⁵ for about 12~16 h. The cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The lyses was centrifuged at 1.5 × 10⁴ cells/well for 15 min at 4 °C and the concentration of these supernatants were assayed by BCA Protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). To separate these proteins, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used, and they were then transferred into nitrocellulose (NC) membranes (PALL, Stevenage, UK). Then, the NC membranes were incubated with specific primary antibodies, including Ki67 (ABclonal Technology, Wuhan, China), STAT3 (ABclonal Technology, Wuhan, China), p-STAT3(Cell Signaling Technology, MA, USA), EXOSC5, GAPDH (Cell Signaling Technology, MA, USA) at 1:1000 overnight at 4 °C. In addition, horseradish peroxidase-conjugated immunoglobulin G (IgG) (Cell Signaling Technology, MA, USA) was used to incubate the membranes at 1:5000 for 1 h at room temperature. Immunoreactive protein bands were detected with an Enhanced Chemiluminescence (ECL) Detection Kit (Amersham, Piscataway, NJ, USA). Image J software was used to analyze the relative levels of proteins normalized to the expression of GAPDH.

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).