Fig. 2: ESCRT targets xenophagolysosomes, and localization of the ESCRT complex is regulated by Rab8A and Rab41.

a Localization of the ESCRT-III component CHMP4B during GAS infection. HeLa cells transfected with CHMP4B-EGFP and mCherry-LC3 were infected with GAS for 4 h, fixed, then stained with DAPI. Shown are representative confocal single slice images of three independent experiments. Scale bar, 2 μm. b HeLa cells were infected with GAS for 4 h, fixed, then immunostained for endogenous LC3 and CHMP4B. Cellular and bacterial DNA were stained with DAPI. Shown confocal images are single slice and representative of three independent experiments. Scale bar, 2 μm. c HeLa cells stably expressing GFP-LC3 were infected with GAS for 4 h, fixed, then immunostained for endogenous CHMP6, VPS28, or SNF8. Cellular and bacterial DNA were stained with DAPI. Shown confocal images are single slice and representative of three independent experiments. Scale bar, 10 μm. d HeLa cells stably expressing GFP-LC3 were infected with GAS in absence or presence of BAPTA-AM (10 μM) for 4 h, fixed, then immunostained for endogenous CHMP4B. Cellular and bacterial DNA were stained with DAPI. Representative confocal single slice images and quantification of CHMP4B recruitment to GcAVs. Scale bar, 10 μm. The proportion of the LC3 signal (GcAVs) overlapping the CHMP4B signal were quantified by Mander’s coefficient M1. Data are individual values and mean ± SEM (n = 50 GcAVs examined over three independent experiments). Unpaired two-tailed t test. e–g HeLa cells stably expressing GFP-LC3 were infected with GAS for the indicated time, fixed, then immunostained for endogenous LAMP1 and CHMP4B. Cellular and bacterial DNA were stained with DAPI. Representative confocal micrographs of single slice (e) and the proportion of the LC3 signal (GcAVs) overlapping the LAMP1 or CHMP4B signal were quantified by Mander’s coefficient M1 (f). Scale bar, 2  μm. Data are mean (circles) ± SEM (shadows) (n = 20 GcAVs examined in each three independent experiments). The percentage of GcAV-positive cells for LAMP1 and CHMP4B were counted using microscopy (g). h–j HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNA, and infected with GAS. Cells were fixed, immunostained for endogenous CHMP4B and stained with DAPI. Representative confocal micrographs of single slice (h), and the proportion of the LC3 signal (GcAVs) overlapping CHMP4B was quantified by Mander’s coefficient M1 (i, j). Scale bar, 10 μm. Data in (i, j) are individual values and the mean ± SEM (30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. k, l HeLa WT, Rab8A KO, Rab41 KO cells stably expressing GFP-LC3 were infected with GAS for 4 h, fixed, and immunostained for endogenous CHMP4B. Representative confocal images of single slice (k) and the proportion of LC3 signal (GcAVs) overlapping CHMP4B quantified by Mander’s coefficient M1 (l). Scale bar, 10 μm. Data in (l) are individual values and the mean ± SEM (30 GcAVs examined over three independent experiments). One-way ANOVA, Dunnett’s test. Source data are provided as a Source Data file.