Fig. 4

The specific autophagic roles of ATG4B and RSV sensitized the autophagy process of the G-cleave LC3B biosensor in MDA-MB-231. A The schematic of the CRISPR/Cas9 sgRNA targeting sequence located in the human ATG4B chromosome. TIDE analysis displayed the B gene editing efficiency and C gene indel spectrum of ATG4B DNA locus in MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc. D Immunoblotting verified the depletion of ATG4B protein expression. E MDA-MB-231 cells expressing the pEGFP-LC3B^pepABLuc were transduced with lentivirus-mediated CRISPR/Cas9 targeting ATG4B or scrambled control (SC) and then treated with either EBSS or serum starvation for 4 h. The luciferase degradation activity (convert to autophagy activity) of the G-cleave LC3B biosensor was assessed. F MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc were treated with or without 100 μM RSV for 4 h. IVIS images were obtained, and quantification of total bioluminescence photon flux was analyzed. G MDA-MB-231 cells expressing pEGFP-LC3B^pepABLuc were treated with RSV at indicated concentrations for 4 h. Luciferase degradation activity (convert to autophagy activity) was determined as described in the materials and methods (n = 3 replicates; student-t test; *, p < 0.01; **, p < 0.01; error bars represent mean ± SE). H MDA-MB-231 cells were treated with 1 to 100 μM RSV for 24 h, with or without 30 μM CQ co-treatment. Cell lysates were collected, and the accumulation of LC3B-II and SQSTM1 was examined by immunoblotting. β-actin was used as a loading control. I MDA-MB-231 cells with pmcherry-EGFP-LC3B expression were treated with 25 μM RSV with or without 30 μM CQ for 24 h. Autophagic flux was determined by observing the formation of EGFP (green), mcherry (red), and merge (yellow/orange) puncta under the confocal microscopy. Scale bar: 10 μm. J 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