Fig 2: Arl8b recruits the HOPS complex to Rab7-PLEKHM1–positive endosomes. (a) Lysates from HEK293T cells treated with control- or Arl8b-siRNA and expressing FLAG-PLEKHM1 were IP with anti-FLAG Abs-resin and IB with indicated antibodies. (b–i) Representative confocal micrographs of HeLa cells treated with either control- or Arl8b-siRNA and expressing FLAG-PLEKHM1 (WT) alone or coexpressed with siRNA resistant Arl8b-tomato and stained for Vps41 or Vps18. (j and k) Colocalization of FLAG-PLEKHM1 with Vps41 or Vps18 was quantified by measuring PC in indicated siRNA-treated HeLa cells (n = 3; 30 cells analyzed per experiment). (l) Western blot of GST-pulldown assay using GST-PLEKHM1 (1–300) as bait incubated with lysates from either WT- or Arl8b KO-HeLa cells with increasing concentration of His-Arl8b protein and immunoblotted (IB) with the indicated antibodies. (m) GST-pulldown assay using semipurified TAP–HOPS complex isolated from HeLa cells incubated with either GST or GST-PLEKHM1 (1–300), His-Arl8b, and excess GTP or GDPßS. (n) Lysates of HEK293T cells treated with either control- or Arl8b-siRNA followed by cotransfection with FLAG-PLEKHM1 and HA-Rab7 were subjected to immunoprecipitation (IP) with anti–HA antibody resin and immunoblotted with the indicated antibodies. Data represent mean ± SEM (****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.

Fig 3: Depletion of HOPS subunits does not alter Rab7 recruitment to SCV.a-f) Representative confocal images of control-, Vps41-, or Vps39-siRNA treated HeLa cells, and subsequently transfected with GFP-Rab7 and infected with DsRed-expressing Salmonella (red). At different times p.i., cells were fixed and stained for LAMP1 (blue). Insets depict higher magnification of the boxed areas showing localization of Rab7 and LAMP1 around the SCVs. Bars: (main) 10 µm; (insets) 5 µm. g) Quantification of Rab7-positive SCVs in control-, Vps41- or Vps39-siRNA treated HeLa cells. Data represent mean ± S.D. over three independent experiments at the indicated time points where ~100 SCVs were counted in each experiment. h and i) Quantification of GFP-Rab7 intensity around the SCVs in control-, Vps41- or Vps39-siRNA treated cells over three independent experiments at the indicated time points p.i. where intensity profile of ~50 SCVs were quantified in each experiment. Data represent mean ± S.E.M. j-m) Representative immunogold EM images of control siRNA (j and k)- or Vps41 siRNA (l and m)-treated HeLa cells infected with Salmonella for 2 hr and transfected with HA-tagged Rab7 and fixed at 10 hr p.i. Cells were processed for immunogold labeling with anti-LAMP1 (10 nm) and anti-HA (15 nm) antibodies. Arrowheads indicate localization of Rab7 (red) and LAMP1 (black) around the SCVs. Bar: 500 nm.

Fig 4: 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.

Fig 5: HOPS subunits are recruited to LAMP1-positive SCVs and SIFs during Salmonella infection.a-d) Representative confocal micrographs of HeLa cells infected with DsRed-expressing Salmonella (red). At different time points post infection (p.i.), cells were fixed and stained for endogenous Vps41 (green) and LAMP1 (blue). Different panels represent a higher magnification of the boxed areas, showing recruitment of Vps41 on SCVs and SIFs (marked by yellow arrowheads). Red arrowheads indicate lysosomal localization of Vps41 in panels (a) and (b). Bars: (main) 10 µm; (insets) 5 µm. e) Time-lapse microscopy of HeLa cells co-transfected with plasmids encoding GFP-Vps41 and untagged-Arl8b, and infected with DsRed-expressing Salmonella (red). Time-lapse series were recorded 9 hr p.i., and still images shown here correspond to S2 Movie. Different panels represent a higher magnification of the boxed area indicating Vps41-positive SIFs emanating from the SCVs showing extension, retraction and bifurcation (white arrowheads). Red arrowheads indicate fusion of Vps41-positive vesicles with SIFs. Bars: (main) 10 µm; (insets) 5 µm. f and g) Quantification of endogenous (f) or HA-tagged Vps41 (g)-positive SCVs at different time points p.i. Data represent percentage of Vps41-postive SCVs scored for ~100 SCVs for each time point. The mean ± S.D. is shown for three independent experiments. h) SCVs were isolated from Salmonella-infected HeLa cells at 3 hr and 8 hr p.i. using sucrose density ultracentrifugation, followed by second round of ultracentrifugation of fractions 8–10 on a ficoll cushion (labeled as SCV). Different fractions were resolved on SDS-PAGE gel and immunoblotted using indicated antibodies. i) Salmonella-modified membranes were isolated from HeLa cells infected with sseF-deficient strain of Salmonella harboring an expression vector with a C-terminal epitope-tagged sseF and its cognate chaperone sscB (sseF/pSseF-HA) at 8 hr p.i. by differential centrifugation. The enriched fraction was further subjected to affinity immunoprecipitation (IP) using anti-HA antibody-conjugated agarose beads or anti-Myc antibody-conjugated agarose beads as a control. The eluted samples were analyzed for presence of effector protein (SseF) and host proteins by Western blotting as indicated.