Extended Data Fig. 5: K48-polyubiquitination of bacteria is key for recruitment of p97.
A. Percentage of p97-positive bacteria in host cells treated with E1-Ub ligase inhibitor PYR41 (10 µM) compared to DMSO control. Cells were treated with PYR41 for 1 h before being infected with different pathogens. At 6-9 h, post-infection, association of intracellular bacteria (stained with DAPI) with p97 (stained with anti-p97 Ab) was assessed by quantifying confocal images. n > 100 events, N = 3. B-D. Confocal micrographs showing the association of p97 (Green) positive SPN (B), STm (C), and GAS (D) (Blue) with K48-Ub (Red) but not with K63-Ub (Magenta). E-G. Confocal images depicting K63-Ub (Magenta) positive but K48-Ub (Red) negative SPN (E), STm (F) and GAS (G) that fail to recruit p97 (Green). Arrows depict events shown in insets besides respective images. For B-G, Bacteria were stained with DAPI, p97 was conjugated to GFP, K48-Ub and K63-Ub were stained with respective primary Ab, followed by Alexa Fluor 555 or Alexa Fluor 633 conjugated secondary Ab. Scale bar, 5 μm. H. Confocal microscopy showcasing diminished K48-Ub levels in OTUB1-HA overexpressing A549 cells compared to WT A549s. K48-Ub (red) and OTUB1-HA (magenta) were detected by staining anti-K48-Ub and anti-HA Ab. Scale Bar, 5 μm. I. Percentage of association of p97 with SPN in FBXW7 downregulated host cells. FBXW7 expression in A549s were knocked down by siFBXW7, followed by infection with SPN. n > 100 events, N = 3. J. Percentage association of STm with p97 in siARIH1 and siRNF213 treated HeLa cells. While AIRH1 is reported to ubiquitinate cytosolic STm with K48-Ub chain types, RNF213 ubiquitinates LPS present on STm outer membrane. n > 100 events, N = 3. Data for A, I, J are means ± SD of respective independent biological replicate (N). Statistical significance was assessed by one-way ANOVA followed by Dunnett’s test in J or two-tailed unpaired student’s t-test (nonparametric) for A, I, with correction for multiple comparisons. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001.
