Ripa solution

After treatment, cells were collected, and the total proteins were extracted using RIPA solution (Servicebio, Wuhan, China) containing a mixture of protease phosphatase inhibitors (Beyotime, Nanjing, China). A BCA protein assay kit was used to measure the total concentrations of proteins according to the manufacturer’s instructions. Twenty micrograms of cellular protein from each group was electro-blotted onto a PVDF membrane (Millipore, Burlington, MA, USA) following separation on 10% SDS-PAGE (Sangon Biotech, Shanghai, China). The membranes were blocked with 5% BSA in 0.1% Tween 20/Tris-buffered saline (TBST) for 2 h at room temperature and then incubated with either anti-BACH1 ((F-9): sc-271211) or anti-HOMX1 (10701-1-AP) antibodies overnight at 4 °C. Afterwards, samples were washed thrice with 0.1% TBST and incubated with secondary antibodies (1:2000) (Beyotime, Nanjing, China) at room temperature for 2 h. The enhanced chemiluminescence kit (Cat No: WBKLS0500) was used to visualize the protein bands.

To perform Western blot analysis, each selected tumor was lysed in RIPA solution (G2002, Servicebio, China) with steel beads in a KZ-III-F high-speed cryogenic tissue grinder (Servicebio, China). We measured the protein amount using a BCA protein quantification kit. In brief, the lysed sample (100 µg) was loaded on 8%–12% polyacrylamide gels and transferred to PVDF membranes. TBST buffer diluted with 5% skimmed milk was then used to block the membranes for 1.5 h, followed by incubation with CYP3A4 and GAPDH antibodies for 8–12 h, respectively. The membranes were then incubated in biotinylated goat antibody IgG for 1 h.