Figure 3.

Chlamydia pecorum-Induced Nuclear Factor Kappa B (NFκB) Activation and Interleukin-6 Secretion are Detectable 24 hpi, Effects not Abolished by Penicillin-Induced Chlamydial Stress. HeLa cells were pre-exposed (A) or not (B,C) to 1 μg/mL cycloheximide (CHX) for 2 h, infected using centrifugation with C. pecorum (Cp) at a multiplicity of infection (MOI) of 1 and incubated until 24 h post infection (hpi) with or without exposure to 1 unit/mL penicillin G (PenG) in the incubation medium. (A) Representative IF micrographs show NFκB p65 (green) and C. pecorum LPS (red) at 1,000x magnification. White and yellow arrows indicate the nuclei of cells with and without NFκB nuclear translocation, respectively. White and yellow asterisks indicate chlamydial inclusions in cells with and without NFκB nuclear translocation, respectively. Scale bar = 10 μm. (B) NFκB activation, specifically of subunit p65, was assayed by an enzyme-linked immunosorbent assay (ELISA)-style assay of whole cell lysates and showed that C. pecorum significantly induced NFκB activation, regardless of PenG-induced chlamydial stress (n = 3, mean ± standard deviation, ^*p < 0.05). (C) Interleukin-6 (IL-6) secretion was assayed by ELISA evaluation of cell culture medium and showed that C. pecorum significantly induced IL-6 secretion, regardless of PenG-induced chlamydial stress (n = 3, mean ± standard deviation, ^*p < 0.05).