Loading buffer

The GSCs were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors for 30 min at 4 °C. Then the supernatants were collected after centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatants were mixed with loading buffer (Boster, Wuhan, China), and equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to PVDF membranes. The PVDF membranes were blocked and incubated with the primary antibodies at 4 °C overnight. The primary antibodies were used at the following dilutions: rabbit anti-β-actin (1:2000; #4970, CST), mouse anti-β-III tubulin (1:1000; #4466, CST), rabbit anti-GFAP (1:1000; BA0056, Boster), mouse anti-sox2 (1:1000; ab171380, Abcam), rabbit anti-CD133 (1:1000; ab19898, Abcam), rabbit anti-H3 (1:1000; #4499, CST), rabbit anti-OCT4 (1:1000; ab18976, Abcam), rabbit anti-acetyl-H3 (1:1000; #06-599, Millipore), rabbit anti-H3K27me3 (1:1000; #9733, CST), mouse anti-H3K9me3 (1:1000; #5327, CST), rabbit anti-EZH2 (1:1000; #5246, CST). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies at room temperature. The antibody labeling was detected using an enhanced chemiluminescence reagent (Merck Millipore). The protein bands were analyzed using ImageJ.

Total protein was extracted using RIPA lysis buffer (including protease inhibitor cocktail). After the protein concentration of each sample was determined by a bicinchoninic acid (BCA) kit, the loading buffer (5×; Boster, Wuhan, China). was added and boiled for 5 min for Western blotting. Equal amounts of protein from each sample were separated using 12.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk at room temperature for 1.5 h and then incubated with a monoclonal rat anti-α7nAChR antibody (1:200; Santa Cruz, CA, United States), and a monoclonal mouse anti-GAPDH antibody (1:10,000; ZenBioScience, China) overnight at 4°C. After incubation with the appropriate HRP-conjugated secondary antibodies at room temperature for 1 h, the membranes were scanned using a Fusion Edge system (Vilber, FR), and the density of the results was quantified using ImageJ software (NIH, Bethesda, MD, United States). The GAPDH were used as internal controls.

Radioimmunoprecipitation assay lysis buffer was used for isolation of proteins from cells (Boster, Wuhan, China), while a bicinchoninic acid kit (Boster, Wuhan, China) was used for determination of protein concentration; the protein samples were heated after being combined with a loading buffer (Boster) at a 5 : 1 ratio. The proteins in the gels were translocated in polyvinylidene difluoride membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was incubated with primary antibodies after blocking with 5% nonfit milk powder for 2 h at room temperature (RT). The primary antibodies were used for the analysis after incubating the proteins with the following secondary antibodies: rabbit anti-TOP2A (1 : 2,500; CST, 12286), rabbit anti-E-cadherin (1 : 10,000) (ab40772), rabbit anti-N-cadherin (1 : 10,000) (ab18203), rabbit anti-vimentin (1 : 5,000; Abcam ab92547), rabbit anti-Snail1 (1 : 1,000; Boster, PB0449), rabbit anti-Snail2 (1 : 2,000; Boster, PB9439), rabbit anti-β-actin (1 : 10,000; Boster, BM0627), and rabbit anti-GAPDH (1 : 2,000; Boster, A00227-1). The secondary antibodies (1 : 10,000) were purchased from Proteintech, China. The experiments were repeated three times. Finally, the amplified chemiluminescent system was used to visually detect the signal.