FIG. 3.

Loss of Rgp activity prevented MV entry into cells. (A) HeLa and IHGE cells were incubated separately with MVs prepared from strains ATCC 33277 (wild strain), KDP129 (Δkgp), KDP133 (ΔrgpA ΔrgpB), and KDP136 (Δkgp ΔrgpA ΔrgpB) at 37°C for 15 min in serum-free medium. After a wash, the cells were incubated in serum-containing medium for 120 min after the addition of MVs. For fluorescence microscopy, the cells were processed for staining for MVs (green) and actin (Alexa Fluor 568-conjugated phalloidin red). (B) Quantification of MV entry into cells. The percentages of mutant MVs that entered in comparison to wild-type MVs are shown. At least 20 cells were analyzed in each experiment, with three independent experiments performed. *, P < 0.01. (C) Wild-type MVs were incubated separately with KYT-1 (10 μM; inhibitor of Rgp) and KYT-36 (10 μM; inhibitor of Kgp) in serum-free DMEM at 37°C for 30 min, after which they were incubated with HeLa cells at 37°C for 15 min. After a wash with DMEM, the cells were incubated for the indicated time periods after addition of MVs in serum-containing medium. Bars (A and C) = 20 μm.