Figure 6: EhC2PK is essential for initiation of phagocytosis.

(a) Cells with and without mentioned constructs were incubated with human erythrocytes for indicated time points and then were fixed and stained with TRITC-Phalloidin. Initiation of phagocytosis was marked by accumulation of actin at the phagocytic cups (marked by an arrow). (b) Fifty cells were randomly selected for each experiment and the number of phagocytic cups present in all the cells were enumerated (blue, normal HM1:MSS; red, C2-domain; green, K179A mutant; purple, antisense). (c) Erythrocyte uptake was determined quantitatively using RBC solubilization assay. The level of expression of cloning indicated that the gene was manipulated by using two different concentrations of G418 (10 and 30 μg ml⁻¹). The experiment was repeated five times independently in triplicates (N=5) with error bars indicating the standard error. The statistical comparisons were carried out using one-way ANOVA test. 'P-values' are mentioned in the figure (the P>0.05 for KD-GFP at 30 μg ml⁻¹, which implies that RBC uptake was not significantly higher than other cell lines maintained at 10 μg ml⁻¹ of G418). (d) Localization of C2-GFP and EhCaBP1 in C2-GFP-expressing cells after 8 min of incubation with RBC. Quantitative analysis of the fluorescent signals of cells undergoing erythrophagocytosis and expressing C2-GFP and EhCaBP1 are shown (pink, cytosol; blue, phagocytic cups). (e) Localization of EhCaBP1 in K179A mutant expressing cells after 8 min of incubation with RBC (pink, cytosol; blue, phagocytic cups). For analysis, five random regions were selected from membrane, cytosol and phagocytic cups and average intensity was computed for each region. This was repeated for four such cells (N=4, bars represent standard error). Relative intensity was calculated by assuming intensity from membrane as 100% for each marker separately (scale bar, 10 μm; DIC, differential interference contrast).