The immunofluorescence colocalisation of GFP-SNX5 with chlamydial inclusion membranes (Figure 6A; Figure 6—figure supplement 1A) imaged on a confocal microscope was measured by Mander’s correlation coefficient of red pixel (EEA1 or mCherry-Rab25) over green pixel (GFP-SNX5) signals, which were determined using ImageJ (https://imagej.nih.gov/ij/) with the JACoP plugin (Bolte and Cordelières, 2006). Punctate structures were automatically counted using ImageJ analyse particle tool across total of 10 cells from two biological replicates. To quantify the effect of PI3K inhibitors on SNX recruitment (Figure 1—figure supplement 2), Z-stacks were captured with a Zeiss 710 confocal laser scanning microscope using a 40x objective. Maximum projections were generated with FIJI (https://fiji.sc/) and Pearson’s correlation coefficients for individual cells determined using the FIJI ‘Coloc 2’ function with Costes threshold regression and 100 Costes randomisations. Co-localization analyses were conducted on two independent experiments from five images per condition each containing at least 20 cells (>100 cells analysed per condition).