Figure 2

Determination of the optimal assay window for capturing LC3-II increase in response to rapamycin. (A) HeLa cells were co-treated for indicated time intervals (0.5–24 h) with rapamycin or vehicle in the presence or absence of 2.5 nM, 10 nM or 2 µM (the latter only tolerated for up to 8 h) BafA. Representative Western blots of LC3-II, p62/SQSTM1 and actin are shown for each treatment time point. (B) Quantification of Western blots from 2 independent experiments (mean ± SEM; linear fit). P62/SQSTM1 levels (left panel) from cells treated with BafA in the absence of rapamycin were quantified, normalized to actin, and subsequently compared to p62/SQSTM1 levels treated with DMSO only (0 nM BafA). LC3-II levels (right panel) were normalized to actin. The LC3-II increase caused by 10 µM Rapamycin over time in the presence of either 0, 2.5, 10 or 2000 nM BafA is compared to the levels induced by the respective BafA-concentrations without rapamycin which was set to 100%.