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. 2019 May 8;9:141. doi: 10.3389/fcimb.2019.00141

Figure 2.

Figure 2

Vi suppresses activation of Cdc42 and Rac-1 in Salmonella infected cells. (A) Hela cells were infected with S. Typhimurium in the absence or presence of Vi and lysed with Triton X-100 (1%). Active Cdc42 and Rac1 (GTP-bound form) were precipitated from cell lysates by affinity pull-down assay using Cdc42/Rac1-binding domain of p21 activated kinase-1 (PAK). PAK-bound proteins and cell lysates were electrophoresed in 10% SDS-polyacrylamide gel and after transferring proteins to a nitrocellulose sheet, immunoblotted with specific antibodies to Cdc42, Rac1, and GST. The blot was developed with ECL. (B) Untreated or Vi-treated (30 min at 37°C) cells were infected with S. Typhimurium (100 MOI) for 30 min. Cells were fixed with paraformaldehyde and incubated with Alexa-fluor 488-labeled Phalloidin to stain filamentous actin. Cells were visualized under a fluorescence microscope (Nikon TE2000) and images were imported into Adobe Photoshop. (C) Uninfected or S. Typhimurium - infected (100 MOI for 30 min) cells were stained for filamentous actin with Alexafluor-488-labeled Phalloidin (green staining). Subsequently, cells were incubated with Vi at 4°C (for prohibitin localization) followed by anti-Vi antibody and Alexafluor 594-labeled anti-mouse Ig Ab (red staining). Images were merged in Adobe Photoshop to see co-localization of F-actin with Vi-bound prohibitin. (D) Cells were treated with Vi or PBS and infected with S. Typhimurium at 100 MOI. The expression of IkB-α, p-JNK and p-PKC-α was evaluated at different time points by western blotting using specific antibodies. The blot was subsequently probed with anti-GAPDH antibody and developed using ECL. (E) Cells (in triplicate) were treated with indicated concentrations of Vi, LPS or equivalent volume of PBS for 30 min before infecting with S. Typhimurium at different MOIs for 30 min. Cells were washed and incubated for another 6 h in serum-free RPMI-1640 supplemented with gentamycin. CXCL8 and IL-6 were determined in culture supernatants by ELISA. (F) Cells (in triplicate) were incubated with Vi (10 μg/ml) or equivalent volume of PBS and stimulated with indicated concentrations of TNF-α. After 6 h, CXCL8 was analyzed in the supernatants by ELISA. Data is representative of 2 independent experiments. P-value *** p < 0.005, NS, not significant.