Induction of flhDC increases intracellular accumulation. a) After flhDC induction, Salmonella (F-Sal) were 33% more motile in aqueous solution than control Salmonella (Sal). b) In aqueous solution, the motile fraction of Salmonella (15-30 μm/s) increased while the non-motile fraction (0-15 um/s) decreased (*, P < 0.05). c) In monolayer culture, Salmonella (green) invaded into MCF7 cells. Salmonella with flagella (control and pflhDC) invaded cells more than non-motile (ΔflgE and ΔflgE+pflhDC) Salmonella. Some ΔflgE+pflhDC Salmonella invaded cells. All Salmonella constitutively expressed GFP. Scale bar is 100 μm. d) Salmonella overexpressing flhDC invaded cells 1.25 times more than control Salmonella (***, P < 0.001). Salmonella with intact flagella (control and pflhDC) invaded cells significantly more than non-flagellated (ΔflgE and ΔflgE+pflhDC) Salmonella (***, P < 0.001). Non-motile ΔflgE+pflhDC Salmonella invaded cells more than ΔflgE Salmonella (**, P <0.01). e) Four strains of Salmonella were transformed with PSSEJ/GFP and Plac/DsRed to identify extracellular (red only) and intracellular (green and red) bacteria. f) The PSSEJ promoter is intracellularly activated. At an early time after invasion (2 hours), Salmonella only express DsRed (top left) and do not express GFP (bottom left). After 18 hours of incubation, intracellular Salmonella express both GFP (bottom right) and DsRed (top right). Scale bar is 100 μm. g) In tumor masses, many of the colonized Salmonella were intracellular. Scale bar is 100 μm. h) Overexpression of flhDC (F-Sal) increased the density of intracellular Salmonella in tumor masses 2.5 fold more than control Salmonella (Sal) at times greater than 29 hours after bacterial administration (*, P < 0.05). i) The average intracellular density of flhDC induced Salmonella was 2.5 fold greater than control Salmonella (*, P < 0.05). j) Induction of flhDC increased intracellular accumulation of F-Sal in medial (0.5 ≤ x ≤ 0.6) and distal (x ≥ 0.8) tumor tissue compared to controls (Sal; *, P < 0.05).