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. Author manuscript; available in PMC: 2017 Oct 20.
Published in final edited form as: Cell. 2016 Oct 20;167(3):670–683.e10. doi: 10.1016/j.cell.2016.09.023

Figure 5. Sca4 acts in trans to target vinculin in the donor cell.

Figure 5

(A) Mixed cell assay schematic, as described in methods. (B) Images of infectious foci detected in the mixed cell assay showing A549-TRT (soluble TagRFP-T, red), R. parkeri (green) and β-catenin (blue). Scale bar, 10 μm. (C) Mixed cell assay results showing percentage of bacteria that spread to recipient cells in a focus. Donor or recipient cells were reverse transfected with control siRNA (NT) or siVCL #1 and infected with WT (Rp-GFP) or sca4::tn. Mean ± SEM, one-way ANOVA: ** p < 0.01 relative to WT + NT in each set. (D) Mixed cell assay results as in (C) except indicated donors or recipients were transduced with FCW2-P2AT (control) or FCW2-P2AT-Ires-Sca4 (Sca4). One-way ANOVA: *** p < 0.001 relative to WT + P2AT in each set. (E and F) Mixed cell assay as in (A, B) except the donor cell was infected with WT (Rp-GFP), sca4::tn or a mixture of sca4::tn and sca2::tn. (E) Image of an infectious focus co-infected with sca2::tn (bright green, open arrow) and sca4::tn (dim green, solid arrow) and counterstained for Rickettsia (blue) and β-catenin (red). Scale bar, 10 μm. Inset, expanded view of donor cell region indicated by solid arrow (inset scale bar, 5 μm). (F) Percentage of bacteria that spread for WT (Rp-GFP), sca4::tn, or sca4::tn in the presence of sca2::tn. No spread was detected for sca2::tn. Mean ± SEM, one-way ANOVA: * p < 0.05 relative to WT. See Figure S2.