SNX9 is recruited to <i>C</i>. <i>trachomatis</i> entry sites.

<p>(A) Quantification of PX-BAR-Myc recruitment to <i>C</i>. <i>trachomatis</i> entry sites. Cultured RPE1 cells were transiently transfected with the indicated PX-BAR-Myc fusion proteins and infected with <i>C</i>. <i>trachomatis</i> LGV2 for 10 min prior to fixation. Fixed cells were stained with an anti-<i>Chlamydia</i> primary antibody and an Alexa Fluor 546-conjugated secondary antibody and an anti-Myc primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Recruitment of PX-BAR-Myc proteins was quantified. ≥ 150 cell-associated bacteria were assessed for recruitment of each PX-BAR-Myc fusion protein, expressed as the average percentage ±SD (n = 2). P-values obtained from Student's unpaired two-tailed t-test comparing recruitment of Myc at 10 minutes post-infection,* P<0.05, ** P<0.01. (B) Localisation of PX-BAR-SNX9 and endogenous SNX9 during infection. Cultured RPE1 cells were infected with <i>C</i>. <i>trachomatis</i> LGV2 for 30 min. Fixed cells were stained with either an anti-Myc or anti-SNX9 primary antibody and an AlexaFluor 488-conjugated secondary antibody. Cells were also stained with an anti-<i>Chlamydia</i> primary antibody and an AlexaFluor 546-conjugated secondary antibody. Images are maximum projections of confocal xy sections of the focal plane containing the EB and the nearest neighbour z-sections (±0.2 μm). Scale bars, 2.5 μm. (C) RPE1 cells were transfected with GFP-SNX9 and were infected with <i>C</i>. <i>trachomatis</i> LGV2 CMTR EBs 18 h later (red). Cells were incubated at 37°C for 15 min prior to imaging. Confocal z-stacks were captured every 10 seconds for 15 min. Panels show maximum projections of confocal z-stacks at 100 s intervals. Scale bar, 7.5 μm.