The PIKfyve inhibitor, YM201636 blocks intracellular but not extracellular replication of Salmonella typhimurium. (A) A431 cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h were infected with RFP-SL1344 for 10 min. The cells were thoroughly washed and cultured for a further 2, 4, 8, 12 or 24 h in the presence of 50 μg/ml gentamycin before being fixed and stained with phalloidin-conjugated to Alexa488 and DAPI. Maximum projections were generated from Z-stacks obtained by confocal microscopy as described in the Materials and methods section. (B) The relative area occupied by RFP-fluorescence was measured for infected cells using ImageJ and associated macros as described in the Materials and methods section. Error bars represent the standard error of the mean. P<0.005. At least 150 cells were counted for each construct and the experiment repeated in triplicate. (C) Colony forming units were calculated as described in the Materials and methods section. (D) SL1344 were cultured in Luria–Bertani broth with 800 nM YM201636 or equivalent volumes of DMSO for the indicated times and OD₆₀₀ readings taken hourly.

PIKfyve activity is required for macropinosome late endosome/lysosome fusion. (A) HEK-FlpIn cells cultured in the presence or 800 nM YM201636 or the equivalent volume of DMSO were pulsed-labelled with dextran (10 000MW) conjugated to tetramethylrhodamine for 5 min prior to fixation in 4% PFA. Macropinosomes, defined as dextran-positive structures >0.5 μm in diameter, were counted using an automated image analysis protocol described previously (Lim et al, 2008). (B) To examine later stage macropinosome maturation, first the late endosomes and lysosomes of FlpIn HEK293 cells were fluorescently labelled by culturing the cells in the presence of 200 μg/ml dextran–Alexa647 (MW 10 000) for 4 h before being thoroughly washed and further cultured in normal growth media for 16 h. Newly formed macropinosomes, labelled by culturing cells in the presence of 100 μg/ml dextran–TR (MW 10 000) for 4 min, were imaged in 4D as described in the Materials and methods section. Presented is a montage of selected frames of macropinosomes in a cell transfected with pPIKfyve-GFP or pPIKfyve(K1831M)-GFP. Total duration and interval were as indicated. The total time taken to internalise dextran–TR (4 min) and initiate imaging is indicated in red. Movies can be viewed in Supplementary data. (C) To quantify the impact PIKfyve-GFP, PIKfyve(K1831M)-GFP, GFP-Rab7, GFP-Rab7(T22N), DMSO, YM201636, GFP-Rab7+YM201636 and GFP-Rab7(Q67L)+YM201636 have upon macropinosome late endosome/lysosome fusion, >100 macropinosomes from at least 50 independent time-lapse videomicroscopy experiments were scored for content mixing. Presented is the proportion of macropinosomes that have acquired content from the late endosomes and lysosomes. Error bars represent the s.d. P<0.005.

YM201636 inhibits Salmonella-containing vacuole (SCV) acidification and activation of the SPI2 virulence locus. (A) A431 cells cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h were infected with PipB-2 × HA SL1344for 10 min. The cells were thoroughly washed and cultured for a further 8 h in the presence of 50 μg/ml gentamycin before being fixed and labelled with monoclonal antibodies against the HA-epitope. (B) A431 cells were cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h and infected with PipB-2 × HA or SseA-2 × HA SL1344 for 2 or 8 h as indicated. Western immunoblotting was conducted as described. (C) A431 cells cultured in the presence of 1 μM LysoTracker Red with 800 nM YM201636 or equivalent volumes of DMSO for 2 h and were then infected GFP-SL1344 for 10 min. The cells were thoroughly washed and cultured in the continued presence of Lysotracker Red and YM201636 or DMSO and 50 μg/ml gentamycin for a further 90 min. Maximum projections of live infected cells were generated from Z-stacks obtained by confocal microscopy as described in the Materials and methods section. (D) A431s cultured in the presence of 800 nM YM201636 or the equivalent volume of DMSO were infected and cultured in gentamycin as described earlier for 24 h before being fixed and examined by resin electron microscopy as described in the Materials and methods section.

YM201636 treatment blocks bacterial degradation in macrophages. (A) Bone marrow-derived macrophages (BMDMs) cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h were infected with RFP-SL1344 for 10 min. The cells were thoroughly washed and cultured for a 24 h in the presence of 50 μg/ml gentamycin before being fixed and stained with phalloidin-conjugated to Alexa488 and DAPI. Maximum projections were generated from Z-stacks obtained by confocal microscopy as described in the Materials and methods section. (B) BMDMs cultured in the presence of 800 nM YM201636 or the equivalent volume of DMSO were infected and cultured in gentamycin as described earlier for 24 h before being fixed and examined by resin electron microscopy as described in the Materials and methods section.

Perturbation of PIKfyve inhibits intracellular Salmonella typhimurium replication. (A) A431 cells transiently transfected with pPIKfyve-GFP or pPIKfyve(K1831M)-GFP. (B) A431 cells were treated with scrambled-, PIKfyve- and Vac14-siRNAs as described in the Materials and methods section for 72 h. (C) A431 cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h. Cells were infected with RFP-SL1344 for 10 min, thoroughly washed and cultured for a further 24 h in the presence of 50 μg/ml gentamycin before being fixed, labelled with monoclonal antibodies against LAMP1 and counterstained with phalloidin conjugated to Alexa488 (B, C) and with DAPI (A–C). Maximum projections were generated from Z-stacks obtained by confocal microscopy as described in the Materials and methods section.

PI(3)P is transiently associated with the macropinosome due to PIKfyve activity. FlpIn HEK293 cells were transiently transfected with (A, B) pEGFP-2 × FYVE_Hrs, (C) pPIKfyve-GFP or (D) pPIKfyve(K1831M)-GFP cultured in the presence of 100 μg/ml dextran–TR (MW 10 000) for 4 min and newly formed dextran–TR-positive macropinosomes imaged in 4D as described in the Materials and methods section. (B) Samples were pretreated with 800 nM YM201636 for 2 h and the experiment run in the continued presence of this concentration of the pharmacological agent. Presented is the average fluorescent intensity of each GFP probe throughout the dextran-positive macropinosome over time. Total duration and interval were as indicated. Three independent replicates are presented. (E) Example frames of each condition is presented as a montage. Note: the presented example is represented by the blue line in each graph.

YM201636 inhibits the maturation of the Salmonella-containing vacuole (SCV) and Salmonella-induced filaments (SIFs) during infection. (A) The late endosomes and lysosomes of A431 cells were fluorescently labelled by culturing the cells in the presence of 200 μg/ml dextran–Alexa488 (MW 10 000) for 16 h before being thoroughly washed and further cultured in normal growth media for 4 h. The cells were cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h and were then infected with RFP-SL1344 for 10 min. The cells were thoroughly washed and cultured for a further 8 h in the presence of 50 μg/ml gentamycin before being fixed and counterstained DAPI. Maximum projections were generated from Z-stacks obtained by confocal microscopy as described in the Materials and methods section. (B, D) A431 cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h were infected with RFP-SL1344 for 10 min. The cells were thoroughly washed and cultured for a further 1 h (B) or 8 h (D) in the presence of 50 μg/ml gentamycin before being fixed, labelled with monoclonal antibodies against LAMP1 and counterstained and DAPI. Maximum projections were generated from Z-stacks obtained via confocal microscopy as described in the Materials and methods section. Arrows indicate SIFs (D). (C) The impact DMSO or YM201636 had on SCV maturation was quantified by counting the number of infected cells that displayed LAMP1-positive SCVs 1 h p.i. Presented is the proportion of SCVs that were LAMP1-positive. Error bars represent the s.d. P<0.005. At least 100 transfected cells were counted for each treatment and the experiment repeated in triplicate. (E) The impact DMSO or YM201636 had upon SIF formation was quantified by counting the number of infected cells that displayed LAMP1-positive SIFs 8 h post-infection. Presented is the percentage of transfected cells that contained at least one SIF. Error bars represent the s. d. P<0.005. At least 100 transfected cells were counted for each construct and the experiment repeated in triplicate.

YM201636 inhibits intracellular replication of Salmonella typhimurium and macrophage bactericidal activity in bone marrow derived macrophages. (A) Bone marrow-derived macrophages (BMDMs) cultured with 800 nM YM201636 or equivalent volumes of DMSO for 2 h were infected with RFP-SL1344 for 10 min and cultured as indicated. Intracellular replication was monitored by measuring the relative area occupied by RFP-fluorescence in (B) infected cells and colony forming unit (C) assayed as described in Figure 2.