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. 2012 Feb 16;11(2):129–139. doi: 10.1016/j.chom.2012.01.006

Figure 4.

Figure 4

Host Arf GEF Promotion of Salmonella-Induced Ruffling and Invasion

(A) Influence of Rac1 and Arf1 depletion on Salmonella invasion and ruffle formation. HeLa cells were transfected with either mock, Rac1, or Arf1 siRNA 72 hr before infection with WT Salmonella (15 min). To quantify Salmonella invasion, washed infected cells were incubated with gentamicin for 2 hr to kill extracellular bacteria, and invasion was quantified by colony counts. To quantify Salmonella with ruffles, fixed infected cells were stained with Alexa Fluor 488-phalloidin to visualize actin. The number of fluorescently labeled bacteria in each field of view was counted and the proportion of those bacteria associated with actin ruffles determined. Asterisks indicate a significant difference from mock (p < 0.05, ANOVA; n = 3). Error bars represent ± SEM. Knockdowns were quantified by qRT-PCR, data not shown.

(B) Representative fluorescence images of Salmonella-induced ruffles in mock- and Arf1-depleted cells from (A). Scale bar 130 μm. Arrows indicate Salmonella-induced ruffles in mock.

(C) Influence of Arf family GEF depletion on Salmonella invasion and ruffle formation. HeLa cells were transfected with either mock or indicated Arf GEF siRNA 72 hr before infection with WT Salmonella (15 min). Quantification of invasion and Salmonella with ruffles was performed as in (A).

(D) Representative fluorescence images of Salmonella-induced ruffles in mock- and ARNO-depleted cells from (C). Scale bar 130 μm. Asterisks indicate a significant difference from mock (p < 0.05, ANOVA; n = 3). See also Figure S2.

(E) ARNO activation of Arf1 during Salmonella invasion. HeLa cells expressing Arf1HA (Arf1) were treated with either DMSO (control) or an inhibitor of the cytohesin family (SecinH3) 1 hr before infection (15 min) with WT Salmonella. Cells were lysed with detergent, and the soluble fractions were incubated with GSTGAT (GAT), then Arf1HA was immunoprecipitated. The fraction of GTP-bound Arf1HA was determined by immunoblotting for coprecipitated GAT with anti-GST antibody and anti-HA as control.

(F) Influence of ARNO inhibition on Salmonella invasion. HeLa cells were treated with either DMSO (control) or an inhibitor of the cytohesin family (SecinH3) 1 hr before infection with WT or ΔE/E2 strains of Salmonella (15 min), and quantification of Salmonella invasion was performed as in (A).