Fig 5. Interaction of dextran-loaded lysosomes with SCVs is impaired upon Vps41 depletion.

a) Schematic illustrating the protocol used for loading of lysosomes with Alexa-Fluor 647-conjugated dextran in HeLa and RAW264.7 cells, prior to infection with GFP-expressing Salmonella. b-e) HeLa cells treated with control siRNA (b) or Vps41 siRNA (c) or RAW264.7 cells transduced with control shRNA (d) or Vps41 shRNA (e) were pre-incubated with Alexa-Fluor 647-conjugated dextran (red) to label lysosomes, followed by infection with GFP-expressing Salmonella (green). Time-lapse series for Alexa-Fluor 647-conjugated dextran loaded and infected cells were recorded at 10 hr p.i., and still images from representative time lapse series are shown (S7–S10 Movies). Different panels represent a higher magnification of the boxed area and the white arrowheads indicate the SCVs. Bars: (main) 10 μm; (insets) 5 μm. f and g) Quantification of Alexa-Fluor 647-conjugated dextran-positive SCVs in control and Vps41 depleted HeLa and RAW264.7 cells fixed at 10 hr p.i. Data represent mean ± S.D. over three independent experiments where ~100 SCVs were counted in each experiment (****, P < 0.0001; Student’s t test). h and i) Quantification of Alexa-Fluor 647-conjugated dextran signal intensity around the SCVs in control and Vps41 depleted HeLa and RAW264.7 cells. Data represent mean ± S.E.M. of signal intensity from three independent experiments at 10 hr p.i. where ~50 SCVs were counted in each experiment.