Minichemi imager

After being treated with IVM, the concentration of the protein of cells was measured using a BCA protein assay kit. Subsequently, equal protein amounts were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, U.S.A.) and electrotransferred to a PDVF membrane (0.22 μm). After the transfer, the nonspecific binding blots were blocked in 5% no-fat milk dissolved in TBST for 1.5 h at 37°C with gentle shaking. Then, the specific primary antibodies were incubated overnight at 4°C, followed by incubation for 1 h with the respective second antibody at room temperature. Signals were detected using MiniChemi Imager (SageCreation, Beijing, China). β-actin was used as the endogenous control.

Total RNA was isolated using TRIzol reagent (TaKaRa), reverse transcribed, and analyzed by quantitative PCR (qPCR) using SYBR Green and an ABI 7300 system (Applied Biosystems, Carlsbad, CA). A complete list of PCR primers is shown in Supplementary Table 1. For protein analysis, we lysed cells or homogenized tissues directly in RIPA buffer, and then centrifuged them at 4 °C for 10 min at 13,200 × g. The resulting supernatant fraction was separated by SDS-PAGE and immunoblotted with the indicated antibodies. Immunoreactivity was detected using Kodak X-OMAT film (Kodak) or a MiniChemi Imager (SageCreation, Beijing, China). The intensities of the bands were quantified using NIH ImageJ software. All the proteins were normalized to the internal control β-ACTIN or GAPDH.

Western blot was used to investigate the expression of autophagy-associated proteins. After treatment, U251 and C6 cells were lysed with RIPA buffer supplemented with PMSF (Beyotime, China) and quantified using the BCA protein concentration assay kit (Beyotime, China). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (Bio-Rad, USA) and electrotransferred to a PVDF (0.22 μm). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4°C. The next day, the membranes were labelled with secondary antibody. Signals were detected using MiniChemi Imager (SageCreation, Beijing, China). β-actin was used as the endogenous control.