FIG 2.

miR-199a-5p regulates Shiga toxin internalization in HeLa cells. (A and B) CM- and miR-199a-5p-transfected cells were incubated with Cy3-StxB (5 μg/ml) on ice for 48 h and then shifted to 37°C for 30 min (A, top, and B) and 60 min (A, bottom) Cells were fixed and labeled with anti-GM130 antibody (A), anti-EEA1 antibody (B), and DAPI (4′,6-diamidino-2-phenylindole) (A and B). Z projections of confocal stacks are shown. Note that miR-199a-5p-overexpressing cells accumulated StxB in peripheral membranes. Scale bar = 15 μm. Magnif, magnifications of areas in dotted boxes. Scale bar = 10 μm. (C) HeLa cells treated with Inh-199a-5p were incubated for 30 min and 60 min with Cy3-StxB as described in the legend to panels A and B. (D and E) Quantification of Manders' coefficients for colocalization of Cy3-StxB localized in the Golgi apparatus (GM130, at 60 min) or early endosomes (EEA1, at 30 min) in experiments whose results are shown in panels A and C or B, respectively. (F) Quantification of Golgi apparatus fragments in control mimic (CM)- and miR-199a-5p-transfected cells in experiments whose results are shown in panels A to C. (G) Western blot analysis of GM130, EEA1, and Vps26A in cells transfected with CM or miR-199a-5p mimics. MW, molecular weight in thousands. Right, quantification of the relative amounts of proteins. The mean values ± SEM from three independent experiments are shown. P values were calculated using the t test. *, P ≤ 0.05.