EhARPC1 is required for phagocytosis.

(A) and (B) Immunoblot analysis of E. histolytica cells(HM-1:IMSS) expressing Tet-O-CAT (TOC) vector alone, or containing the cloned antisense EhARPC1 (AS) gene or sense EhARPC1 (S) in the presence and absence of tetracycline (30μg/ml). EhCaBP1 was used as an internal control. (C) Erythrocyte uptake assay was performed in over expressing (sense) and down regulated cell line (antisense) with vector alone as control. The assay was performed in the presence and the absence of tetracycline. The experiments were repeated independently three times. One-way ANOVA test was used for statistical comparisons. (D) Cells overexpressing either sense or antisense constructs of EhARPC1 were incubated with erythrocytes for the indicated times at 37°C. Cells were then fixed and stained for EhARPC1 (Alexa-488) and actin with TRITC-Phalloidin. Phagocytic cups are marked by arrowhead, star marks the just closed cup before scission and yellow arrowheads show attached RBCs at the site of phagocytosis (E) and (F) Quantitative analysis of phagocytic cups and phagosomes in over expressing (EhARPC1 sense), down regulating (EhARPC1 antisense) and Wild type HM1: MSS cell-lines, was carried out by randomly selecting 25 cells (in triplicates) and counting the number of phagocytic cups and phagosomes present in these cells. One-way ANOVA test was used for statistical comparisons. “One black star” p-value≤0.05, “Two black star” p-value≤0.005, “Three black star” p-value≤0.0005.