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Figure 1


Fig. 1. The SH4 domain of Leishmania HASPB induces plasma membrane blebbing. (A) Confocal microscopy analysis of HASPB-expressing CHO cells. Stable CHO cell lines expressing the SH4 domain of Leishmania HASPB fused to GFP (N18-HASPB-GFP) (a-d) or expressing a HASPB mutant with the myristoyl acceptor glycin at position 2 replaced by alanine (N18-{Delta}myr-HASPB-GFP) (e-h) under control of a dox-dependent promoter were cultured in the presence of 1 µg/ml dox to induce transgene expression. 24 hours post-induction, cells were analyzed by confocal microscopy either live (a,e) or following fixation and staining with TRITC-labelled phalloidin to visualize cellular F-actin (fixed; b-d,f-h). Presented are representative mid-cell z-sections. Scale bars, 10 µm. (B) PM blebbing induced by non-fusion full-length HASPB. Shown are confocal micrographs of live cells expressing: full-length HASPB fused to GFP (a), full-length non-fusion HASPB (b), GFP (c) or a palmitoylation-deficient mutant of N18-HASPB fused to GFP (N18-{Delta}pal-HASPB-GFP; d). All GFP fusion proteins were expressed stably in CHO cell lines, whereas full-length non-fusion HASPB was transiently co-expressed in CHO cells with actin-RFP. Panels display the distribution of GFP fusion proteins with the exception of b, in which actin-RFP is shown. Scale bars, 10 µm. (C) PM blebbing as a function of N18-HASPB-GFP expression levels. N18-HASPB-GFP CHO cells were cultured for 24 hours with the indicated concentrations of dox, fixed and analyzed for PM blebbing by microscopy. Shown is the overall percentage of cells displaying PM blebs with the black and grey columns indicating the fraction of blebbing cells with high (more than five blebs per cell) or low (less than five blebs per cell) blebbing activity, respectively. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition. (D) Quantification of the blebbing efficiency of the cells depicted in selected panels in A and B. Values are the arithmetic means of at least three independent experiments experiments+s.d. In each experiment, over 100 cells were counted per condition.