D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and (A-C) fixed and treated with an anti‐ubiquitin antibody, or (D) lysed, fixed and analyzed by flow cytometry. LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (B) The percentage of D. discoideum with intact LCVs was assessed for the following number of cells: JR32, n = 132; JR32 ∆plcABC, n = 606; Corby, n = 265; Corby ∆plaACD, n = 357. (C) The percentage of D. discoideum harboring ubiquitinated L. pneumophila was assessed for the following number of cells: JR32, n = 100; JR32 ∆plcABC, n = 602; Corby, n = 281; Corby ∆plaACD, n = 389. (D) Quantification of GFP-positive bacteria of the L. pneumophila strains by flow cytometry and FlowJo software. Data shown are means and SEM from (B, C) three biological replicates and (D) a technical triplicate (two-tailed Student's t test; *P < 0.05, and **P < 0.01). 