Figure 6

EGFR is required for C. trachomatis -induced calcium mobilization. (A) and (B) Calcium mobilization induced by C. trachomatis infection. HeLa cells treated with control siRNA, EGFR siRNA or PDGFRβ siRNA were infected with C. trachomatis for 2.5 h or 5 h and stained with Fluo-4 AM for visualization of calcium (Ca²⁺) by fluorescence microscopy. The fluorescence intensity of calcium staining from three independent experiments was quantified using ImageJ (B). (C) Inclusion development and organization of F-actin at the chlamydial inclusion periphery. HeLa cells were pre-treated with BAPTA/AM (a calcium chelator) for 1h followed by infection with C. trachomatis for 24 h, fixed, and processed for confocal microscopy. Data from three independent experiments were analyzed for number and size of inclusions that were significantly reduced in BAPTA/AM treated cells (left panel). F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-chlamydial LPS antibody (red) (right panel). Note the diffused assembly of F-actin at the inclusion periphery (arrow). (D) Inclusion development in post infection BAPTA/AM treated cells. HeLa cells were infected with C. trachomatis for 24 h. BAPTA/AM or control DMSO was added at 2 or 5 hpi. Under all conditions the total time for C. trachomatis infection was 24 h followed by fixing, and processing for confocal microscopy. F-actin was detected with Alexa Fluor 488-phalloidin (green) and chlamydial inclusions were detected using anti-C. trachomatis EB antibody (red). Data is representative of two independent experiments. Note the impaired inclusion development in BAPTA/AM treated cells in comparison to the DMSO treated cells. F-actin staining for HeLa cells with BAPTA/AM treatment but without C. trachomatis infection is also shown in Additional file 2: Figure S12. Scale bar - 10 μm.