RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry
Figure 3
PDGFRβ contributes to C. trachomatis binding.
(A) HeLa cells were transfected with control siRNA or siRNA targeting Abl kinase, PDGFRβ, or EGFR and subsequently infected with C. trachomatis for 1 hour. Cells were fixed and analyzed by inside out staining for the total cell-associated bacteria (black bars) and the internalization efficiency (light grey bars). The data are expressed as the percentage of cell-associated bacteria (which includes internalized and surface-associated bacteria) or as the internalization efficiency (the percentage of internalized EBs/total cell-associated EBs). In each case, the data are normalized to control siRNA. ***p<0.001 for PDGFRβ RNAi-treated cells compared to control RNAi-treated cells (ANOVA). The percentage of cell-associated bacteria was significantly decreased only in cells depleted of PDGFRβ, but not Abl kinase or EGFR. (B) Western blot analysis of siRNA-treated samples. C, control; A, Abl; β, PDGFRβ; E, EGFR. Control, Abl and PDGFRβ samples were run on the same blot. GAPDH was used as a loading control. The efficiency of protein depletion compared to control siRNA-treated cells is indicated to the right of each panel. (C) HeLa cells were pretreated with control IgG or a neutralizing antibody to PDGFRβ or EGFR at the indicated concentrations, subsequently infected with C. trachomatis for 1 hour, and analyzed for total cell-associated EBs. Data for PDGFRβ or EGFR neutralizing antibody was normalized to the same concentration of each isotype matched IgG control and set at 100%. ***p<0.001 or **p<0.01 for cells pretreated with PDGFRβ neutralizing antibody compared to the same concentration of isotype matched IgG control (ANOVA). The percentage of cell-associated bacteria was significantly decreased only in cells treated with a neutralizing antibody to PDGFRβ but not EGFR. The small decrease in binding observed upon pretreatment with 2 ug/ml of EGFR neutralizing antibody is not statistically significant.