R. parkeri interacts with the ER. (A) Percentage of bacteria interacting with the ER structures in B. The mean ± SEM of three independent experiments (large outlined circles) is superimposed over the raw data (small unlined circles) representing the percentage per FOV (>2,000 bacteria counted per independent experiment, data are colored by experiment). (B) Live-cell imaging snapshots at 28 hpi of WT R. parkeri (magenta, RpBFP) in vacuole-like (arrowhead) or protrusive (arrow) ER structures (cyan, mCherry-Sec61β). Scale bar, 5 µm; inset, 2.5 µm. (C) Relative frequency of bacteria interacting with the ER in B that are forming protrusive or vacuole-like structures at 28 or 48 hpi. Error bars indicate SEM from three independent experiments (>80 bacteria counted per independent experiment). (D) Relative frequency of bacteria interacting with the ER that display an actin tail at 48 hpi (as in E). Data presented are mean ± SD. Approximately 10 FOVs were imaged per strain for each replicate (n = 2) and >100 bacteria interacting with the ER were quantified. (E) From part D, live-cell imaging snapshots at 48 hpi of SIR-actin (yellow, open arrowhead) and RpBFP (magenta) in vacuole-like (arrowhead) or protrusive (arrow) ER structures (cyan, BiP-mNeonGreen-KDEL). Scale bar, 5 µm. (F and G) Live-cell imaging snapshots of vacuole-like rickettsia–ER structures (arrowhead) over time. Scale bar, 2.5 µm. Seconds, s; minutes, min.