Extended Data Fig. 4: Cytosolic pathogens are targeted by p97.
A-C. Confocal micrographs showing p97 (Green) positive SPN (A), STm (B), and GAS (C) (Blue) that are devoid of membrane marker FM4-64 (Red). D-F. Confocal images depicting FM4-64 (Red) positive SPN (D), STm (E) and GAS (F) that fails to recruit p97 (Green). Arrows depict events shown in insets besides respective images. Scale bar, 5 μm. G-H. Percentage population of p97-positive bacteria that are marked with or without membrane marker FM4-64 (G) or damaged endosome marker Gal8 (H). n > 50 events. Data are means ± SD of N = 3, independent biological replicates. A549 and Hela cells were infected with SPN (MOI ~ 25), GAS (MOI ~ 2) for 9 h and STm (MOI ~ 50) for 6 h, respectively. Bacteria were stained with DAPI, membranes were marked with FM4-64, while p97 and Gal8 were detected with respective primary Ab, followed by Alexa Fluor 488 or Alexa Fluor 555 conjugated secondary Ab. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s test in G, H, with correction for multiple comparisons. ***P < 0.001, ns, non-significant.
