EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica
Fig 5
EhP3 is essential for phagocytosis.
(A) (i) Expression analysis of EhP3 in sense and antisense cell lines. 50 μg of the lysate prepared from E. histolytica cells expressing either Tet-O-CAT (TOC) vector alone, antisense EhP3 (AS) or sense EhP3 (S) gene grown in presence and absence of tetracycline (10, 30μg/ml) were loaded in respective lanes. The lanes were probed with anti-EhP3 antibody for expression analysis and anti-EhCaBP1 antibody was taken as equal loading control. (ii) Erythrocyte uptake assay in sense and antisense cell lines. E. histolytica cells carrying the above mentioned constructs were incubated with RBCs for the indicated time points and assessed for RBC uptake by spectrophotometric analysis. The experiments were repeated independently three times in duplicate with error bars indicating the standard error. The statistical comparisons were carried out using oneway AnoVA test (p-value 0.00005). (B) (i) and (ii) Visualization of phagocytic events in sense and antisense cell lines. Cells incubated with RBCs were fixed and stained for actin with TRITC-Phalloidin, or with anti-EhP3 antibody followed by Alexa-488. Image panels in the left indicate TRITC-Phalloidin stained multiple number of EhP3 sense cells (i) and antisense cells (ii), (Scale bar, 5 μm; DIC, differential interference contrast). Image panels in the right indicate cells stained with anti-EhP3 antibody and TRITC-Phalloidin respectively, (Scale bar, 10 μm; DIC, differential interference contrast). (C) (i) and (ii) Quantitative determination of phagocytic events (phagocytic cups and phagosomes) observed in above indicated cell lines. Number of phagocytic cups and phagosomes were counted in randomly selected 50 cells (in triplicates) at denoted time points. One-way ANOVA test was used for statistical comparisons. “*” p-value 0.05, “**” p-value 0.005, “***” p-value 0.0005, “****” p-value 0.00005.