Alexa fluor 488 or 594 conjugated secondary antibodies

Immunofluorescence assay according the following steps: first the cells fixed in 4% paraformaldehyde for 0.5 hour at room temperature, PBS buffer washing for three times, and blocked with 5% donkey serum for 2 hours at room temperature, then incubated with anti-Nrf2 and anti-PRDX5 antibodies (Abcam) at 4°C overnight. PBS buffer washing for three times, the cells then were incubated with Alexa Fluor 488 or 594 conjugated secondary antibodies (Abcam) for 1 hour at room temperature. DAPI (Sigma-Aldrich) was used for nuclear staining. After adding anti-fluorescence quenching agent seal, the cells were visualized by the fluorescence microscopy (Leica DMR 3000; Leica Microsystem, Bensheim, Germany).

Auranofin (AF) was purchased from Abcam Inc. (Cambridge, MA, USA). The 3-(4,5- Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), N-acetylcysteine (NAC), dimethylsulfoxide (DMSO) and 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO,USA). SB203580 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Erlotinib, SP600125, FR180204, and Bromodeoxyuridine (BrdU) were purchased from Selleck Chemicals LLC (Houston, TX, USA). Primary antibodies and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), except BrdU primary antibody, which was purchased from Proteintech (Wuhan, Hubei, China). Alexa Fluor^®488- or 594-conjugated secondary antibodies were from Abcam Inc. (Cambridge, MA, USA).

Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4μm interval were cut from each tissue, and stained with hematoxylin and eosin (H&E), or via immunohistochemistry (IHC) or immunofluorescence (IF). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 minutes. Tissues were blocked with 0.5% BSA in PBS for 30 minutes and incubated with primary antibodies against: SMAD4, CD3 (Santa Cruz), CD45, or PD-L1 (Cell Signaling) at 1:50–1:200 overnight at 4°C. Slides were developed using HRP-conjugated secondary antibodies followed by DAB substrate/buffer (DAKO).
For immunofluorescence, slides were heated via pressure cooker in DAKO retrieval buffer and tissues blocked with 0.5% BSA in PBS for 1 hour at room temperature. Sections were exposed to primary antibodies against CK19 (University of Iowa Hybridoma Bank), E-Cadherin (Cell Signaling), CD3, (Santa Cruz), or IFNγ (abcam) at 1:50–1:200 overnight at 4°C. Slides were developed using AlexaFluor 488- or 594-conjugated secondary antibodies (1:200–1:1,000, abcam), mounted in DAPI-containing media (Santa Cruz Biotechnology), exposed to DAPI, FITC, and Texas Red filters.