Fig. 6

IFN-γ-induced Irgm1 ^−/− mouse embryonic fibroblasts (MEFs) show autophagic flux impairment. a WT, Irgm1 ^−/−, Irgm3 ^−/−, and Irgm1/Irgm3 ^−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, 40 μg/mL rapamycin (RAP) for 2 hours or left untreated. Samples were lysed and equal sample amounts were analyzed by SDS-PAGE/western blot. Western blots were probed with anti-LC3 and anti-actin antibody. b Quantification of 6a, representing ratios of LC3-II and actin band intensities for each sample. Results of four independent experiments are shown. Asterisks mark samples that were not included in the specific experiment. c Wild type (WT) and Irgm1 ^−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, 40 μg/mL RAP for 2 hours, or left untreated. Cells were fixed and stained for LC3 and LAMP1. Representative microscopic images of LC3 and LAMP1 co-localization are shown. Arrows point at the LC3 structures magnified at the end of each panel in the following array: upper left: LC3, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. d Quantification of 6c and S7, showing percent of LC3 structures co-localizing with LAMP1; 50 cells per sample were quantified and the results of two independent experiments are shown. e WT and Irgm1 ^−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours or left untreated and transfected with EGFP-LC3. Cells were fixed and stained for LAMP1, Irga6 (165/3), and Irgb10. Irga6 and Irgb10 were detected with the same secondary antibody (Donkey anti-rabbit Alexa555), so they both appear in the same channel. Representative microscopic images of LC3, LAMP1, and Irga6/Irgb10 co-localization are shown. Arrows point at the LC3 structures shown in enlargement at the end of each panel in the following array: upper left: LC3, upper right: Irga6 and Irgb10, lower left: LAMP1, lower right: Merge of Irga6, Irgb10, and LC3. Scale bars represent 10 μM. f Quantification of 6e, showing percent of LC3 structures co-localizing with LAMP1 and percent of LC3 structures co-localizing with Irga6, Irgb10, and LAMP1; 50 cells per sample were quantified and the results of two independent experiments are shown