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. 2020 May 12;31(6):107638. doi: 10.1016/j.celrep.2020.107638

Figure 4.

Figure 4

CDC42-Activated F-Actin Nucleation by the Arp2/3 Complex Is Essential for Cocoon Formation

(A–D) Knockdown of CDC42 or the Arp2/3 complex subunit ARPC3 inhibits cocoon assembly. Representative time lapses monitoring actin rearrangements during early Shigella WT infection are presented for the scramble siRNA-treated control (Scr; A), for CDC42 (B; KD 87.1%), and for ARPC3 knockdown (C; KD 95.6%) (scales: 5 μm, 1.05 μm z stack; see Figure S3F). (D) Quantitative analysis of actin cocoon assembly shows strongly reduced cocoon formation (no siRNA [HeLa], n = 374 bacteria; siRNA Scr, n = 481; siRNA CDC42, n = 464; siRNA ARPC3, n = 573).

(E) The ability of CDC42 to cycle between an active and inactive state is required for efficient cytosolic access of Shigella. T17N, inactive, GDP-bound mutant; G12V, active, GTP-bound mutant (n = 1,123 total rupture events; WT, n = 373; T17N, n = 375; G12V, n = 375).

(F) Neither RNAi-mediated RAC1 knockdown nor its inhibition by the RAC1 inhibitor ETH1864 alters actin cocoon assembly (siRNA Scr, n = 386; siRNA RAC1, n = 409; DMSO, n = 385; ETH1864, n = 328).

Depicted are mean values ± SD of 3 independent experiments; p < 0.05 is significant compared with control: ∗∗∗∗p < 0.0001; ns, not significant. See Figures 3A, S3, S5, and S6.