Fig. 2: FAM134B is targeted by Salmonella to block ER-phagy.
From: The ER-phagy receptor FAM134B is targeted by Salmonella Typhimurium to promote infection
A WT, FAM134B KO, TEX264 KO and FIP200 KO HEK293A cells stably expressing the ss-RFP-GFP-KDEL were either kept in nutrient rich media, starved for AA for 6 h or starved for 3 h followed by ST infection for 3 h in AA starvation media. ER-phagy was measured by ss-RFP-GFP-KDEL processing. Actin was used as a loading control. AA, amino acids; ST, Salmonella Typhimurium. B Normalized Free RFP-Actin ratio from cells in (A). Data are presented as mean values ± SD of seven biological experiments. ANOVA, *P < 0.05; ***P < 0.001; ns, no significance. C Cells from (A) were either AA starved for 6 h, or AA starved for 3 h, followed by ST infection for 3 h in AA starvation media, fixed and imaged by confocal microscopy. NR, Nutrient Rich. Representative images are shown. ss-RFP-GFP-KDEL, yellow; Free RFP, red. Scale bar 5 μm. D Average number of RFP puncta per cell from (C) were quantified. Data are presented as mean values ± SD of 5 biological experiments. The averages were calculated from a minimum of 100 cells. ANOVA, ***P < 0.001. E WT, FAM134B KO, TEX264 KO and FAM134B KO cells transfected with WT FAM134B were infected with ST. Bacterial content was determined through a colony-forming unit (CFU). Data are presented as mean values ± SD of three biological experiments. ANOVA, **P < 0.01; ***P < 0.001. F FAM134B KO cells transfected with either FAM134B WT or FAM134B LIR mutant were kept in nutrient rich media, starved for AA for 6 h or starved for 3 h followed by ST infection for 3 h in AA starvation media. ER-phagy was measured by ss-RFP-GFP-KDEL processing. Actin was used as a loading control. G Cells from (F) were fixed and imaged by confocal microscopy. Representative images are shown. ss-RFP-GFP-KDEL, yellow; Free RFP, red; Fam134B, far red. Scale bar 5 μm. H Normalized Free RFP-Actin ratio from cells in (F) and average number of RFP puncta per cell from (G) were quantified. Data are presented as mean values ± SD of five biological experiments. The average number of RFP puncta was calculated from a minimum of 100 cells. ANOVA, ***P < 0.001; ns, no significance. Source data are provided as a Source Data file.
