Figure 3.

Effect of APEX2 construct expression during the primary infection on plasmid stability. HeLa cells were infected with Ctr L2 transformed (detailed on the x-axis) as in Figure 1, and construct expression was induced with the indicated concentrations of anhydrotetracycline (aTc) at 7 h post-infection. At 24 h post-infection duplicate coverslips were either fixed to determine construct expression or lysed to release infectious chlamydial organisms to infect a fresh monolayer (secondary infection) in the presence of penicillin to monitor retention of plasmid. Chlamydia that did not retain the expression plasmid would become susceptible to penicillin and become morphologically aberrant. This morphological form is readily distinguished from normal chlamydial development forms by indirect immunofluorescence microscopy. Treating Ctr L2 transformants of IncA_TM-APEX2 or APEX2 with 0.1 nM aTc did not result in construct expression, and, therefore, these conditions were eliminated from analysis. 100 inclusions per coverslip were determined to contain either normal or aberrant Chlamydia (see Supplemental Figure 3). This assay had three biological replicates within two independent experiments. Data are expressed as mean and standard error of the mean of the percentage of inclusions containing aberrant organisms, were graphed, and statistically analyzed using GraphPad Prism 7.0 software. Data were analyzed for statistical significance using two-way ANOVA with Tukey's multiple comparisons post-test, and are indicated with ^**** for p < 0.0001, and ^*** for p < 0.001.