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Fig. S1. Quantification of CLASP-GFP distribution in T. cruzi-infected cells. Using MetaMorph® Imaging software, a line (shown in white) was drawn perpendicular to the internalized parasites in CLASP1-GFP, CLASP2-GFP or cytosolic GFP expressing HFF (left panels). A Linescan graph was generated in MetaMorph® to reveal pixel intensity values for GFPs along the line (right panels).

Fig. S2. Ca2+-regulated lysosome exocytosis is not impaired in CLASP1-depleted cells. Release of β-hexosaminidase in streptolysin-O-permeabilized HFF in the presence of Ca2+ in CLASP1-depleted and non-targeting control-transfected (siControl) cells.

Fig. S3. Silencing of CLASP1 inhibits T. cruzi trypomastigote invasion.

A. Relative expression of CLASP1 mRNA in HeLa cells measured by quantitative RT-PCR 48 h post transfection with a non-targeting siRNA pool (siControl) and the following CLASP1-targeting reagents: Dharmacon siGENOME SMARTpool M-00683-01-0005 containing four siRNA duplexes (SmartPools 1 and 2; two different lots). Individual siRNAs from the original pool: (#1) D-006831-01-005, (#2) D-006831-02-005, (#3) D-006831-04-005, (#4) D-006831-17-005 and ON-TARGETplus SMARTpool L-006831-00-0005.

B. Relative T. cruzi infection normalized to infection of control-transfected cells.

Fig. S4. Comparison of the impact of CLASP1- and CLASP2-depletion on T. cruzi infection and the actin cytoskeleton.

A. siRNA-mediated silencing of CLASP1, CLASP2 or both transcripts in HeLa cells 48 h post transfection as measured by quantitative RT-PCR for CLASP1 mRNA (black bars) or CLASP2 mRNA (grey bars) normalized to cellular GAPDH levels. Relative expression compared with CLASP1 in cells transfected with non-targeting siRNA (C) the value of which is arbitrarily set at 1.0.

B. Relative number of intracellular parasites in siControl (C) and in CLASP1- CLASP2- or CLASP1/2-silenced cells in a 20 min invasion assay.

C. Scoring parasite vacuoles for host LAMP1 association in CLASP1- CLASP2- or CLASP1/2-silenced cells. Cells were pulsed with T. cruzi for 20 min and vacuole maturation allowed to progress for 40 min (total) before examining relative lysosome association with the vacuole.

D. Fluorescent images of aldehyde-fixed HeLa cells following transfection with the specific siRNAs indicated for 48 h and stained with FITC-phalloidin and DAPI. Scale bar = 20 μm.

E. Positioning of T. cruzi vacuoles in HFF relative to the host cell nucleus in pulse-chase infections of cells transfected with a non-targeting siRNA pool (siControl) or siRNAs targeting CLASP1 (siCLASP1), CLASP2 (siCLASP2) or both (siCLASP1/2). Cells were pulsed with T. cruzi trypomastigotes for 15 min followed by a ‘chase’ in medium for 1.5 h. Graphical representation of quantitative data showing relative number of parasites that exhibit juxtanuclear localization for 50 infected cells. Data are represented as the mean ± SD for three coverslips.

Fig. S5. Toxoplasma gondii infection is not affected by CLASP1 depletion. HeLa cells transfected with negative control or CLASP1 siRNAs were infected with β-galactosidase-expressing T. gondii tachyzoites (RH strain) and relative β-galactosidase in cell monolayers measured at 72 h post infection.

Fig. S6. Perinuclear clustering of lysosomes triggered by alkaline pH is impaired in CLASP1-depleted cells. Three representative images of HeLa cells transfected with non-targeting siRNA (siControl) or siRNA pools targeting CLASP1 (siCLASP1), CLASP2 (siCLASP2) or both (siCLASP1/2) following a 15 min exposure to Ringer's-BSA pH 7.2 or pH 7.6 to induce perinuclear localization of lysosomes. Aldehyde-fixed and permeabilized cells were stained with mouse anti-LAMP1 and anti-mouse Alexa594 (red) and counterstained with DAPI to visualize nuclei (blue).

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