Figure 7.

The ATG9A^F382A-expressing mammalian cells display an autophagy defect. (A) For the localization of ATG9A, U2OS ATG9A^−/− cells were transfected with a plasmid expressing GFP (GFP), GFP-ATG9A (WT) or GFP-ATG9A^F382A (F382A) for 24 h before subjecting them to nutrient starvation in EBSS for 2 h. Cells were then fixed and processed for immunofluorescence using anti-ATG16L1 antibodies. Dashed squares indicate the position of the insets while arrows denote colocalizations. Scale bar: 5 μm. (B) Quantification of the experiment shown in panel A. Co-localization between GFP-ATG9A and ATG16L1 was assessed using the Pearson’s correlation coefficient. (C) U2OS ATG9A^−/− cells were transfected as in panel A and treated with 200 nM BafA1 for 2 h, or starved in EBSS in the presence or the absence of 200 nM BafA1 for 2 h. Cells were then fixed and processed for immunofluorescence with anti-LC3 antibodies. Representative images of the EBSS+BafA1 are shown. Scale bar: 5 μm. (D) Quantification of the experiment described in panel C. The number of LC3 puncta per cell was analyzed using automatic counting and data are represented as fold increase compared to the WT fed condition. Values are means of three independent experiments ±SD. At least 30 cells were analyzed per conditions. Asterisks and violet bar indicate significant differences. (E) U2OS ATG9A^−/− cells were transfected and treated as in panel C, but then lysed and processed for western blot analysis using antibodies against GFP, LC3 and actin. ACTA1/actin served as the loading control. (F) Quantification of the autophagic flux in the experiment described in panel E by determining the LC3-II:LC3-I ratio for each sample. Data are means of three independent experiments ±SD. Asterisks indicate significant differences.