Wolbachia levels negatively correlate with host translation levels in JW18 cells.

(A) Illustration of Click-iT method that fluorescently labels newly synthesized proteins in host cells as a measure of translation level. JW18 cells were incubated with a modified methionine called HPG containing an alkyne or whole testes were incubated with a modified puromycin containing an alkyne. In each case these alkyne-containing reagents were incorporated into newly synthesized proteins during incubation. Thereafter, samples were processed for the ‘click’ reaction by adding an azide-tagged fluorophore (purple) which reacted with the incorporated alkynes to label new proteins fluorescently (purple). The fluorescent intensity is used as a measure of translation level within the host cell. This assay is combined with Wolbachia-detecting 23s rRNA FISH assay (green). (B) JW18 cells labeled by Click-iT HPG (purple), DAPI (white), and Wolbachia-detecting 23s rRNA FISH assay (green). Scale bars represent 10μm. (C) Quantification of HPG Click-iT assay fluorescent signal for protein synthesis within individual cells of Wolbachia-free JW18DOX cell line compared to Wolbachia-infected JW18 cell line showing a significant 23.6% decrease in translation level in JW18 cell line (Non-parametric, Mann Whitney, p<0.0001). (D) Quantification of HPG Click-iT assay fluorescent signal for protein synthesis within individual cells of the JW18 population. Each dot represents a single cell. Cells were imaged at 63x to capture the whole cell using Z-stack imaging. The integrated density of the fluorescent signal for each cell was calculated using Fiji and signal was normalized for the size of each cell. We categorized cells according to Wolbachia infection level: no Wolbachia, low Wolbachia (1–10 bacteria), or medium-high Wolbachia (11 bacteria or more). Translation level is significantly decreased in cells that contain Wolbachia. The higher the level of Wolbachia, the lower the translation level. (p<0.001). All bars represent median. See S15 Fig for correlation analysis.