SEPT2 inactivation impairs the entry of InlB-coated beads.
(A) Quantification of InlB-induced phagocytosis in control (CTRL) and SEPT2-depleted HeLa cells. For internalization assays, beads were analyzed by immunofluorescence (see Materials and Methods) for being extracellular or intracellular in at least 50 host cells counted for each of n≥2 separate experiments per siRNA treatment. The left graph depicts the total % of internalized 1 µm InlB-coated beads at 5 minutes post-incubation of siRNA-treated cells, calculated from the (total number of internalized beads) / (total number of cell-associated beads) ×100. Results were analyzed for statistical significance using the z-test for percentages. The right graph depicts the average number of internalized 1 µm InlB-coated beads per cell at 5 minutes post-incubation of siRNA-treated cells. Graphed data represent this average value ±SEM, where results were analyzed for statistical significance using the two sample z-test. (B) Representative scanning electron microscopy images of siRNA-treated HeLa cells to depict the membrane surface of control (CTRL) and SEPT2-depleted cells. Magnification = 1500×, where scale bars indicate 10 µm. (C) Uptake of InlB-beads by siRNA-treated cells. Control (CTRL) and SEPT2-depleted HeLa cells were incubated with 1 µm InlB-beads for 5 minutes, and cells were processed for scanning electron microscopy. Depicted here are representative images of InlB-beads internalized (i.e. CTRL cells), or only adherent (i.e. SEPT2-depleted cells), for siRNA treatments. Hollow arrows indicate internalized beads, solid white arrows indicate extracellular adherent beads. Magnification = 15000×, where scale bars indicate 1 µm.