Filopodial capture is significantly attenuated in SNX9^-/- cells.

<p>(A) Non-infected HAP1 WT and SNX9^-/- cells have similar F-actin organisation and filopodia number. Cultured HAP1 WT and SNX9 ^-/- cells were fixed and stained with rhodamine-phalloidin to visualise F-actin. Scale bars, 10 μm. The number of filopodia per cell was quantified in maximum projections of confocal xy sections with the criteria that the cells quantified has at least three ‘edges’ not adherent to a neighbouring cell, and that filopodial projections were ≥1 μm in length. 15 cells were assessed for WT and SNX9^-/- cells and the results are expressed as the average ±SD (n = 3). P-values obtained from Student's unpaired two-tailed t-test, * P<0.05. (B) The numbers of EBs in association with filopodia during entry is decreased in the HAP1 SNX9^-/- cells after infection by sedimentation or egress. Cultured HAP1 WT and SNX9^-/- cells were infected with <i>C</i>. <i>trachomati</i>s LGV2 for 2h. At 2 h post-infection cells fixed with 1% PFA and stained with an anti-<i>Chlamydia</i> primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cells were permeablised and stained again using anti-<i>Chlamydia</i> primary antibody and an Alexa Fluor 633-conjugated secondary antibody. Intracellular bacteria were labelled with only one fluorophore (Alexa Fluor 633). Extracellular EBs in association with filopodia, ring, cup/tail, or other F-actin structures were scored, and expressed as the relative percentage of EBs in association with each structure ±SD for ≥150 cell-associated bacteria (n = 3). P-values obtained from Student's unpaired two-tailed t-test,* P<0.05, ‘ns’ not significant. Data from one representative experiment is shown for egress as infection is asynchronous therefore it is inappropriate to cross-compare data from individual experiment for statistical analysis. Scale bars, 10 μm. (C) Entry efficiency is decreased in the SNX9^-/- cells. Cultured HAP1 WT and SNX9^-/- cells were infected by egress with <i>C</i>. <i>trachomati</i>s LGV2. At 2 h post-infection the cells fixed with 1% PFA and stained as in ‘(B)’. Representative images are maximum projections of confocal xy sections. Scale bars, 10 μm. Entry efficiency was calculated in HAP1 SNX9^-/- cells relative to HAP1 WT cells. ≥ 200 cell-associated bacteria were assessed and expressed as the average ±SD (n = 3). P-values obtained from Student's unpaired two-tailed t-test, ** P<0.01.