Fig. 2

EBSS and serum starvation induce autophagy in MDA-MB-231 cells expressing the G-cleave LC3 biosensor. A MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc were subjected to either Earle balanced salt solution (EBSS) or serum starvation for varying durations as indicated. B MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc were subjected to EBSS or serum starvation in the presence or absence of 30 μM CQ for 24 h. Cell lysates were collected and subjected to immunoblotting analysis to assess the levels of autophagic lipidation (LC3B) and degradation (SQSTM1). GAPDH and β-actin were used as loading controls. C MDA-MB-231 cells expressing pmCherry-EGFP-LC3B were treated with EBSS or serum starvation medium in the presence or absence of 30 μM CQ for 24 h to determine autophagic flux. The formation of EGFP (green), mCherry (red), and merge (yellow/orange) puncta was observed by the confocal microscopy. Scale bar: 10 μm. D Quantitate analysis of mCherry (red), EGFP (green), and merged (yellow/orange) puncta per cell are represented as means ± SEM in 20 to 30 cells in three independent experiments