Gapdh

Total protein from shPOLQ and shCtrl SK-HEP-1 and BEL-7404 cells were subjected to western blot analysis. Briefly, after bathed in boiling water for 10 min, the total cellular proteins were subjected to SDS-PAGE (10%). After transferring to polyvinylidene difluoride (PVDF) membranes, blots were incubated with 5% BSA (Gibco) in Tris-buffered saline containing 0.5% Tween 20 for 60 min and incubated overnight at 4 °C on a rocker with the following primary antibodies: anti-POLQ (biorbyt, # orb48495), anti-mTOR (abcam, # ab2732), anti-p-mTOR (abcam, # ab137133), anti-CCND1 (CST, # 2978), anti-Bcl-2 (abcam, # ab196495), anti-c-Myc (CST, # 5605) and anti-GAPDH (Bioworld, # AP0063). Following washing three times with TBST for 5 min, the plots were then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG polyclonal secondary antibody (1:3000, Beyotime, # A0216) at room temperature for 1 h. Amersham’s ECL + plusTM western blotting system kit was used for color developing. Signals were detected with enhanced chemiluminescence, using GAPDH as the internal standard (Kodak), and analyzed by ImageJ software.

To determine the time-dependent effect of H₂O₂ on Nox4 protein expression in HUVECs, the cells were treated with H₂O₂ (400 μM) for different time intervals (30 min, 1, 2, or 4 h). To determine the BYHWD effects on H₂O₂-induced Nox4 protein expression, the cells were divided into control, H₂O₂ (400 μM), BYHWD (30 mg/ml), and H₂O₂ + BYHWD (10, 20 and 30 mg/ml) groups. The cells were pretreated with various known concentrations of BYHWD (i.e., 10, 20, or 30 mg/ml) for 6 h. Bicinchoninic acid (BCA) protein assay kits (Sigma, China) were used for the quantification of protein extracts. Proteins were separated in equal amounts (50 μg per lane) using gel electrophoresis technique on SDS-polyacrylamide (10%) gel and transferred to polyvinylidene difluoride Millipore membrane (Thermo Scientific, USA). Membranes were incubated overnight at 4°C in a ratio of 1 : 1000with goat anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma) or primary goat anti-Nox4 (Abcam, USA) followed by incubation for 3 h at room temperature with secondary rabbit antibody conjugated to HRP (1 : 3000, Cell Signaling, USA). Then, employing higher chemiluminescence and X-ray film (Kodak, USA), the signals were visualized and data were presented as the ratio of Nox4 to GAPDH.