Fig. 4: RAB10 dephosphorylation is required for STm invasion.

a Representative images of WT or RAB10 KO Henle 407 cells transfected with the indicated constructs (PM-mCherry with empty vector or indicated PM-RAB10 construct), infected with WT BFP-STm for 30 min and labeled with CellMask. b Quantifications of a and Supplementary Fig. 5. Scission is represented as the percent of bacteria in SCVs. The total number of bacteria that were positive for PM-mCherry at the invasion site was quantified and the proportion of these bacteria that were negative for CellMask was determined. These bacteria were in a sealed compartment that was considered an SCV and the number was used to represent the invasion efficiency at 30 min p.i. (c and d) Representative images (c) and quantifications (d) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells, or LRRK2 KO Henle 407 cells complemented with LRRK2 WT or mutant expression. The cells were infected with WT STm and cell lysates were collected and immunoblotted at 30 min p.i. for phospho-RAB10 (T73), total RAB10, or ß-actin (loading control). e WT or LRRK2 KO Henle 407 cells transfected with indicated constructs (PM-mCherry with empty vector or indicated LRRK2 construct), and then infected with WT BFP-STm SL1344 strain for 30 min and labeled with CellMask. PM scission was quantified as in c. Representative images are shown in Supplementary Fig. 6a, b. Data shown are means ± S.D. for three independent experiments. At least 25 independent cells (b and e) for each condition in each experiment were scored for PM scission. P values were calculated using two-way ANOVA. In b and e P values were calculated between the KOs (RAB10 KO or LRRK2 KO) and their respective WT controls. Scale bars, 10 μm. Source data are provided as a Source Data file.