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. 2024 Apr 10;27(5):109623. doi: 10.1016/j.isci.2024.109623

Figure 4.

Figure 4

ATG9B modulates actin rearrangement in HeLa cells

(A) Schematic representation of the RhoGTPase pathway and the pathways blocked by the respective inhibitors.

(B and C) Treatment of ATG9B siRNA-treated HeLa cells with ROCK inhibitor (Y27632), PAK inhibitor (G5555), LIMK inhibitor LIMKi 3, or Arp2/3 complex inhibitor (CK666) for 2 h. Cells were fixed and immunostained for F-actin (magenta), and cellular DNA was stained with DAPI (cyan). Asterisks indicate actin aggregation. Representative microscopic images (B) and quantification of cell percentage with F-actin aggregation (C).

(D) Co-IP between FLAG-tagged ATG9B and mClover-tagged Rho GTPase proteins.

(E) Immunoblotting of Rho-binding domain or p21-binding domain agarose-isolated active RhoA, Rac1, or Cdc42 from whole cell lysates (WCL) obtained from ATG9B siRNA-treated HeLa cells.

(F) Immunoblotting analysis of ATG9B siRNA-treated HeLa cells with the indicated antibodies.Data in c (n = 100 cells per condition) 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.