The percentage of aldolase A-positive inclusions increases over time and is not due to nutrient deprivation or sustained de novo bacterial protein synthesis. HeLa cells expressing 3×FLAG-tagged AldoA were infected with the mCherry CtL2 strain then fixed and stained with anti-FLAG antibodies at the indicated time postinfection. (A) Quantification of the percentage of inclusions positive for 3×FLAG-AldoA at 8, 18, 24, and 32 h postinfection in the absence or presence of 1 μg/ml cycloheximide (CHX). (B) Quantification of the percentage of 3×FLAG-AldoA-positive inclusions at 18, 24, or 24 h postinfection with medium replacement at 18 h postinfection (24 h*). (C) Quantification of the percentage of 3×FLAG-AldoA positive inclusions at 24 h postinfection (–CHL) or after an additional 8 h of incubation with 40 μg/ml chloramphenicol and fixed at 32 h postinfection (+CHL). Data show the means and SD of a combination of three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not statistically significant (Student t test or one-way ANOVA with multiple comparisons).