C. trachomatis L2 infection of HeLa cells and inclusion area measurements.

HeLa cells seeded at 1.0 × 10⁵ cells/well in a 24-well plate containing a glass coverslip were infected with C. trachomatis L2 CT813-FLAG, IncF-FLAG, CT226-FLAG, or CT483-FLAG transformed strains or wild-type C. trachomatis L2. At 7 hpi, the C. trachomatis L2 strains were induced with 1 (C. trachomatis L2 IncF-FLAG only), 5, or 20 nM aTc. As a control, wild-type C. trachomatis L2 was treated, or not, with 20 nM aTc. Coverslips were methanol fixed at 36 hpi and stained for immunofluorescence to determine inclusion area. A minimum of 100 inclusions per condition were measured using ImageJ. The inclusion area (μm²) and standard deviation were plotted using GraphPad Prism 8.4.0 for three biological replicates (Fig. 1A and Fig. S1A). These data were analyzed for statistical significance using a one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test.

Additional controls including C. trachomatis L2 transformed with CT813 (no epitope tag) and C. trachomatis L2 transformed with pBOMB-mCherry, a vector that constitutively expresses mCherry (i.e., empty vector control) (44), were also used. For these experiments, HeLa cells seeded on coverslips were infected with C. trachomatis L2 CT813 and induced, or not, at 7 hpi with 20 nM aTc. Coverslips were methanol fixed at 36 hpi and stained for immunofluorescence to observe construct expression (anti-CT813 antibody; red), IncA (green), or DNA (DAPI; blue) (Fig. S1B). For the empty vector control, C. trachomatis-infected HeLa cells were fixed at 24 hpi using 3.25% formaldehyde and 0.025% glutaraldehyde to preserve the mCherry and stained for immunofluorescence to observe IncA (green) or DNA (DAPI; blue). Coverslips were imaged at ×63 magnification.