Stubrfp sensgfp lc3 assay

The StubRFP-SensGFP-LC3 assay was performed according to manufacturer’s instruction (Genechem, Shanghai China). Briefly, the U87 cells (5 × 10⁴ cells/mL) seeded on a culture dish were incubated with the StubRFP-SensGFP-LC3B lentivirus (MOI = 5 × 10⁶ TU/mL). New medium was changed after 12 h of incubation. At transinfection 72 h, the cells were treated with silibinin, and then incubated with lysotracker blue or mitotracker deep red (Invitrogen company, Eugene, OR). Finally, the cells were observed by a researcher who was blinded to group allocation with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

The StubRFP-SensGFP-LC3 assay was performed according to the manufacturer’s instructions (GeneChem, Shanghai, China). Briefly, U87 cells (5 × 10⁴ cells/mL) seeded in a culture dish were incubated with StubRFP-SensGFP-LC3B lentivirus (MOI = 5 × 10⁶ TU/mL). The medium was changed after 12 h of incubation. After infection for 72 h, the cells were treated with TMZ at the indicated dosage for the indicated time and then treated with MitoTracker deep red (Invitrogen company, Eugene, OR). Finally, the cells were observed under a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan).
Then, U87 cells seeded in a culture dish were incubated in the presence or absence of TMZ at the indicated dosage for the indicated times before they were treated with 100 nmol/L MitoTracker red (Invitrogen Company, Eugene, OR) for 30 min at 37 °C before fixation in ethanol. After the nonspecific antibody binding sites were blocked, the cells were incubated with anti-BNIP3 (1:100) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) for 1 h. Finally, the cells were visualized under a laser scanning confocal microscope (Olympus FV3000, Tokyo, Japan).