Fig 2: The formation of PTM-MT scaffolds during infection is enhanced in the absence of RhoA. (A and B) Control and RhoA KO HeLa cells were infected with WT C. trachomatis L2 (MOI of 2) and transfected with empty vector or myc-RhoAWT DNA at 4 hpi. Cells were fixed at 24 hpi and labeled with anti-detyrosinated α-tubulin (Dt α-tubulin) (A, green), anti-acetylated α-tubulin (Ac α-tubulin) (B, green), anti-myc (red), and anti-MOMP (blue) antibodies. Scale bar, 30 μm. The graphs represent the average percentage of inclusions with Dt α-tubulin (A) or Ac α-tubulin (B) scaffolds normalized to infected control HeLa cells. A minimum of 100 inclusions was counted for each condition per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (C) Control and RhoA KO HeLa cells were infected with InaC-FLAG-overexpressing C. trachomatis L2 (MOI of 5) and treated with 5 ng/ml anhydrotetracycline (aTc) 4 hpi to induce InaC-FLAG expression. Cells were lysed at 24 hpi, and InaC-FLAG was immunoprecipitated using an anti-FLAG antibody. ARF1 binding was assessed with anti-ARF1 antibody. Lysates were analyzed as a control. Anti-RhoA antibody confirms the loss of RhoA in the RhoA KO. HSP70 served as a loading control. Results are representative of 2 independent experiments. (D) Control and RhoA KO HeLa cells were infected with InaC-FLAG-overexpressing C. trachomatis L2 (MOI of 5) and transfected with ARF1T31N-HA or ARF1Q71L-HA at 4 hpi. InaC-FLAG expression was induced with 5 ng/ml aTc 4 hpi. Cells were lysed at 24 hpi, and InaC-FLAG was immunoprecipitated using anti-FLAG antibody. ARF1 binding was assessed with anti-HA antibody. Lysates were analyzed as a control. Anti-RhoA antibody confirms the loss of RhoA in the RhoA KO. HSP70 served as a loading control. Results are representative of 2 independent experiments.