Figure 2

EGFR is important for bacterial attachment and inclusion development. (A) and (B) Effect of EGFR inhibition on number and size of inclusions. (A) HeLa cells treated with Erlotinib , Cetuximab, EGFR siRNA or PDGFRβ siRNA were infected with C. trachomatis for 24 h, fixed, analyzed by confocal microscopy and ImageJ software. Data from three independent experiments are expressed as percentage of total number of inclusions (black bar) or inclusion size (gray bar) in comparison to the control. (B) MEFs EGFR^+/+ and MEFs EGFR^-/- were infected and processed as in (A). Data are from three independent experiments. (C) and (D) Effect of EGFR inhibition on chlamydial attachment and entry into the host cell. HeLa cells treated with Erlotinib, EGFR siRNA and PDGFRβ siRNA (C) or Cetuximab (D), were infected with C. trachomatis for 2.5 h and inside out staining was performed to differentially stain both external and internalized C. trachomatis. Data from three independent experiments are expressed as number of cell-associated bacteria (external + internalized C. trachomatis) and internalized C. trachomatis per infected host cell. (E) and (F) Levels of chlamydial Hsp60 antigen. HeLa cells treated with Erlotinib, Cetuximab, EGFR siRNA or PDGFRβ siRNA were infected with C. trachomatis for 2.5 h and Western blotting was performed with anti-chlamydial Hsp60 antibody. Quantification of the Western blots is from three independent experiments. (F). β-actin was used as loading control. (G) and (H) Transmission electron micrographs of HeLa cells infected with C. trachomatis. HeLa cells treated with Erlotinib (G) and Cetuximab (H) with respective controls, were infected with C. trachomatis for 24 h and fixed for transmission electron microscopy. Note the typical large inclusions in the control chlamydia infected cells. In cells treated with Erlotinib and Cetuximab the inclusions are smaller and less mature. Scale Bar = 2 μm.