Fig 2: Vaccinia-Mediated Cell Contraction Is Not Dependent on RhoA(A) Phase-contrast images of HeLa cells infected with the F11-VK virus and quantification of their average area (blue line). The F11-VK infection was performed at the same time as WR (black line) and ?F11L (red line) taken from Figure 1A.(B) Images of HeLa cells infected with WR treated with non-targeting (NT) control or RhoA siRNA, together with quantification of their average area. The immunoblot shows the efficiency of RhoA knockdown, and a-tubulin represents a loading control.(C) Representative images of HeLa cells infected with WR in the presence or absence of the C3 Rho inhibitor, together with quantification of their average area.All scale bars, 5 µm. Error bars represent the SEM from three independent experiments, in which a total of 60 (A), 80 (B), and 100 (C) cells were analyzed. See also Figure S3.

Fig 3: RhoC Is Required for Vaccinia-Induced Cell Contraction(A) Immunoblot analysis of glutathione pull-downs on WR-infected HeLa cell lysates reveals that GST-F11 interacts with GFP-tagged RhoA, RhoC, RhoD, RhoE, and RhoF.(B) Quantification of the area of HeLa cells treated with the indicated siRNA at 3 hr 40 min post infection with WR.(C) Quantification of the area of HeLa cells expressing GFP or GFP-tagged wild-type (WT) or T19N (dominant-negative) RhoC 3 hr 40 min after infection with WR (left) or ?F11L (right).(D) Images showing the association of GFP-RhoC with the plasma membrane in cells infected with WR for 3 hr 40 min. The time is indicated in seconds and mCherry provides a volume marker (see Movie S4). Scale bar, 5 µm.(E) Quantification of WR-infected HeLa cell area at 3 hr 40 min post infection in the presence or absence of C3, 72 hr after treatment with the indicated siRNA.(F) Normalized cell area of WR-infected HeLa cells at 3 hr 40 min post infection, 72 hr after treatment with the indicated siRNA.Error bars in graphs represent the SEM from three independent experiments, in which a total of 90 (B), 60 (C), and 80 (E and F) cells were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. See also Figure S4.

Fig 4: RhoD Negatively Regulates Vaccinia-Stimulated Cell Contraction(A) Quantification of the area of HeLa cells treated with the indicated siRNA for 72 hr at 3 hr 40 min post infection with the ?F11L virus.(B) Quantification of the change HeLa cell area after treatment with RhoD siRNA and infection with the ?F11L virus.(C) Images showing the association of GFP-RhoD with the plasma membrane in cells infected with WR for 3 hr 40 min. The time is indicated in seconds and mCherry provides a volume marker (see Movie S5). Scale bar, 5 µm.(D) Left graph shows the quantification of the area of HeLa cells, treated with RhoD siRNA and expressing GFP-tagged wild-type (WT), G26V (constitutively active), or T31N (dominant-negative) RhoD and infected with ?F11L at 3 hr 40 min post infection. Right graph shows the area of HeLa cells expressing GFP-tagged G26V (constitutively active) or T31N (dominant-negative) RhoD and infected with WR for 3 hr 40 min.(E) The area of RhoD-depleted HeLa cells, with or without H1152 (top) or C3 (bottom) treatment, and infected with ?F11L at 3 hr 40 min post infection.(F) The area of HeLa cells depleted of RhoD or RhoD and RhoC and infected with ?F11L at 3 hr 40 min post infection. The immunoblot shows the efficiency of RhoD and RhoC knockdown, and actin represents a loading control.(G) The graph shows the level of GTP-bound RhoC in non-infected U-2 OS cells expressing GFP, GFP-RhoD G26V, or T31N mutant. Error bars represent SEM from four independent experiments. The immunoblot illustrates a representative assay.Error bars in cell-area graphs represent the SEM from three independent experiments, in which a total of 90 (A and B), 70 (E), and 60 (D and F) cells were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Fig 5: Pak6 Inhibits RhoC-Driven Cell Contraction(A) Quantification of the area of HeLa cells treated for 72 hr with the indicated siRNA and infected with the ?F11L virus for 3 hr 40 min.(B) Images showing the subcellular localization of GFP-tagged Pak4, Pak5, and Pak6 during WR infection. Only GFP-Pak6 is associated with the plasma membrane, and mCherry provides a volume marker (see Movie S6). Scale bar, 5 µm.(C) Quantification of the area of WR- or ?F11L-infected HeLa cells treated with Pak6 siRNA and expressing GFP-tagged WT or kinase-dead (KD) Pak6.(D) The area of Pak6 depleted HeLa cells infected with ?F11L at 3 hr 40 min post infection and treated with H1152, C3, or RhoC siRNA.(E) The area of WR-infected HeLa cells depleted of RhoD or Pak6 and expressing GFP-tagged Pak6 or RhoD, respectively.Error bars in graphs represent the SEM from three independent experiments, in which a total of 90 (A and D) or 60 (C and E) cells were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. See also Figure S6.