Figure 3.

ATG9B KD cells form F-actin aggregates and control invasion by several bacterial species

(A) Immunoblotting analysis of ATG9A or ATG9B siRNA-treated HeLa cells using the indicated antibodies.

(B–D) Immunostaining of fixed ATG9B siRNA-treated HeLa cells for p-Paxillin (green) and F-actin (magenta). Cellular and bacterial DNA was stained with DAPI (cyan). Asterisks indicate actin aggregation. Representative microscopic images (B) and quantification of cells for the number of p-Paxillin particles (C) and percentage with F-actin aggregation (D).

(E) Immunoblot analysis of F-actin and G-actin in ATG9B siRNA-treated HeLa cells with quantification using ImageJ/Fiji.

(F–H) Internalization of the indicated bacteria by ATG9B siRNA-treated HeLa cells infected at an MOI of 10. The relative percent internalization was normalized with data from non-targeting control siRNA-treated cells (colony-forming units [CFU] recovered at 2 h post-infection [hpi]/CFUs at 1 hpi).

(I and J) ATG9B siRNA-treated HeLa cells infected with GAS for 2 h and stained with anti-GAS antibody (green) and F-actin (red). Cellular and bacterial DNA was stained with DAPI (cyan). Representative microscopic images (I) and quantification of cells containing F-actin positive GAS (J). Data in C (n = 200 cells per condition), D (n = 100 cells per condition), and E–H represent the mean ± SEM from five independent experiments. Scale bar, 10 μm. One-way ANOVA was performed and p values were calculated using Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.