Caspase 3

2′,7′-Dichlorofluorescein diacetate (DCF-DA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, the antibody raised against HO-1 (1 : 500), BLVRB (1 : 500), and Gclm (1 : 1000) from Proteintech Group, Inc. (Chicago, IL, USA); Nrf2 (WB, 1 : 1000, IF, 1 : 200) and Gsta3 (1 : 500) from Abcam (Cambridge, MA, USA); SRXN1 (1 : 500) from Bioss (Beijing, China); Caspase 3 (WB, 1 : 1000) from Abmart (Shanghai, China); and β-actin (WB, 1 : 2000) from OriGene Technologies, Inc. (Rockville, MD, USA). Alexa Fluor 546 anti-mouse secondary Ab (IF:1 : 2000, A-11030), goat anti-mouse IgG (H + L) peroxidase conjugated (WB: 1 : 20000, 31430), and goat anti-rabbit IgG (H + L) peroxidase conjugated (WB: 1 : 20000, 31460) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Total protein was extracted from cell lines using lysis buffer (Thermo Fisher Scientific), containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China), and quantified using the Bradford method. Next, 40 µg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8%), and the separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies against: TRAF4 (H2818, 1:100, Santa Cruz Biotechnology), Eg5 (1:1000), Smurf2 (F0641, 1:100, Santa Cruz Biotechnology), HA (TA180128S, 1:1000, Origene), α-tubulin (ab52866, 1:1000, Abcam), Caspase-3 (M005851F, 1:1000, Abmart), Bcl-2 (T40056F, 1:1000, Abmart), Bax (T40051F, 1:1000, Abmart), Ki67 (550609, 1:1000, BD Pharmingen), GAPDH (AF0006, 1:2000, Beyotime) and β-actin (1:2000, Beyotime). Next, the membranes were incubated with secondary HRP-conjugated antibody, anti-mouse immunoglobulin G (IgG), or anti-rabbit immunoglobulin (1:2000, Santa Cruz Biotechnology) at 37°C for 2h. Finally, antibody binding wasvisualizedusing electro-chemiluminescence (Thermo Fisher Scientific), and quantified using ImageJ (National Institute of Health, Bethesda, MD, USA).

We used Ishikawa and SNGM cells during the logarithmic growth period to extract total proteins, and protein content determined by BCA Protein Quantification Kit. Take a protein sample and heat it at 100 °C for 3 min to make the protein fully denatured. After separating the mixed protein sample by using polypropylene gel (10% separation gel, 5% concentrated gel), it was placed onto a polyvinylidene difluoride (PVDF) membrane and sealed with 5% skim milk powder (2.5 g non-fat powdered milk + 50 ml Tris-buffered saline with Tween 20). Incubate these membranes with antibodies at 4 °C for 12–16 h: KIF23 (1:2000; Affinity), p-ERK (1:1000; Beyotime), ERK (1:1000; Beyotime), p-AKT (1:1000; ABclonal), AKT (1:5000; ABclonal), p-PI3K (1:1000; ABclonal), PI3K (1:2000; Bioworld), BCL-2 (1:1000; Beyotime), BAX (1:1000; Beyotime), Caspase-3 (1:1000; Abmart), and GAPDH (1:1000; Beyotime). Subsequently, the sample was coupled with peroxidase-coupled anti-rabbit IgG antibody (1:5000; Beyotime, China) for 2 h under room temperature. ECL enhanced chemiluminescence kit (BOSTER, USA) Using a supersensitive ECL chemiluminescence ready-to-use substrate (BOSTER, USA) to visualize the strip on the Bio-RAD machine.