Anti caspase 3

Western blot was performed as described in previous study^{17 (link)}. Rabbit anti-HPV16-L1, anti-HPV16-E6, anti-HPV16-E7 (R&D, USA), Rabbit anti-P53, anti-Bax, anti-Caspase9, anti-Caspase3, anti-cleaved-Caspase3 and anti-β-actin (Affinity, China) were used as the primary antibodies. After being incubated with secondary-HRP-antibodies (Promega, USA), the protein levels were detected with BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and the densities of the specific bands were quantified with an imaging densitometer (Tanon, China).

OSCC cell lines (HSC-3 and HN12) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The cells were routinely cultured in high glucose DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, United States) and 1% antibiotics at 37°C in a 5% CO₂ incubator. The primary antibodies anti-PERK, anti-p-PERK, anti-eIF2α, anti-p-eIF2α were purchased from Cell Signaling Technology (1:1000, United States). The primary antibodies anti-ATF4, anti-caspase-3, anti-cleaved-caspase-3, anti-Bcl2, anti-Bax were obtained from Affinity (1:500, United States). Antibodies α-tubulin, anti-HSP60, anti-IF1, anti-VDAC, and anti-CHOP were obtained from Abcam (1:1000, Cambridge, MA). The OXPHOS complexes were obtained from Thermo Fisher Scientific (1:1000, United States). Antibody p-Ser was obtained from Abcam (1:1000, AP0932, China). Goat anti-rabbit/mouse secondary antibody was purchased from ZSGB-BIO (Beijing, China). Nebivolol was purchased from Selleck (Shanghai, China). 4-Phenylbutyrate (4-PBA) was purchased from Sigma-Aldrich (St. Louis, MO, United States). MitoTracker Red was obtained from Solarbio (Beijing, China).

CD4⁺ T cells lysate was prepared by lysing in RIPA buffer containing protease inhibitors. Protein concentrations were estimated (BCA protein assay kit). After that, the protein samples were loaded onto each lane by SDS-PAGE for electrophoresis and transferred to 0.45 μM PVDF membrane (Millipore, MA, USA). Next, PVDF membranes were blocked with 5% skimmed milk in TBS-Tween for 60 min, followed by incubation with antibody at 4°C for 12-16 h. The following primary antibodies are as follows: anti-mTOR (Cat#AF6308, dilution rate 1 : 1000), anti-P-mTOR (Cat#AF3308, 1 : 1000), anti-P-p70s6k (Cat#AF3228, 1 : 1000), anti-p70s6k (Cat#AF6226, 1 : 1000), anti-4EBP (Cat#AF6432, 1 : 1000), anti-caspase-3 (Cat#AF6311, 1 : 1000), anti-Bcl-2 (Cat#AF6139, 1 : 1000), anti-Bax (Cat#AF0120, 1 : 1000), anti-GRP78 (Cat#AF5366, 1 : 1000), anti-CIRP (Cat#DF2643, 1 : 1000), anti-CHOP (Cat#DF6025, 1 : 1000), anti-CIRP (Cat#AF5366, 1 : 1000), and anti-actin-β (Cat#AF7018, 1 : 3000) were purchased from Affinity Biosciences (Jiangsu, China). Anti-P-4EBP (anti-eIF4EBP1, ab27792) was from Abcam (CA, USA). After washing with TBS-Tween 3 times, the membranes were incubated with a goat anti-rabbit IgG antibody (1 : 5000, Affinity Biosciences) at 25°C for 1 h, and the chemiluminescence signals were detected using an electrochemiluminescence detection system. Band densities were quantified by the ImageJ software.