Fig. 5

Impaired autophagic flux after Ate1 disruption. WT, Ate1 KO, and Ate1 KO^Ate1-1 cells were treated with 20 μM CQ and/or STV for 6 and 24 h. a Immunoblot analysis of LC3B levels. Ate1 and total protein markers were used as Ate1 deletion and loading controls, respectively. All immunoblot samples were obtained from the same experiment and were processed simultaneously. b Quantification of LC3-II relative to total protein marker under serum-rich conditions. Quantification represents the mean ± SE of four independent experiments. c, d Quantification of the LC3-II/LC3-I ratio under serum-rich (b) or serum-starved (c) conditions. Quantifications represent the mean ± SE of three independent experiments. e Immunofluorescence analysis of autophagic vacuoles in WT, Ate1 KO, and Ate1 KO^Ate1-1 cells treated with 20 μM CQ and/or STV for 6 h. Immunostaining was performed using LC3B (red) and Ate1 (green) antibodies and visualized with confocal microscopy. Nuclei (blue) were stained with Hoechst dye. f Quantification of autophagic vacuoles. The number of vacuoles was determined using the “Find Maxima” plug-in of the Fiji ImageJ software. n=10 cells. Statistical significance was assessed using a two-sided unpaired t test. * and # represent differences between cell types and treatments, respectively. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, ns=nonsignificant. Scale bar: 10 μm