FIG. 7.

Axl facilitates macropinocytosis into SNB19 cells. (A) Ability of Axl RNAi to inhibit dextran but not Tfr and CTb uptake. SNB19 cells were transfected with 200 pmol of a nonspecific siRNA control (Block-It) or 200 pmol of a human Axl-specific siRNA. At 48 h following RNAi transfection, cells were incubated for 1 h with Cy5-Tfr, Cy5-CTb, or FITC-dextran. After 1 h, cells were analyzed by flow cytometry for uptake of labeled conjugates. (B) Macropinocytosis inhibitors do not inhibit FIV-ZEBOV transduction when Axl is knocked down by RNAi in SNB19 cells. SNB19 cells were transfected with Axl siRNA or control siRNA (Block-It; Invitrogen). At 48 h posttransfection, cells were treated for 1 h with the indicated amounts of macropinocytosis inhibitors. Treated cells were transduced with FIV-ZEBOVΔO (MOI, 0.005) for an additional 6 h in the presence of drugs. The medium was refreshed after 6 h with medium not containing drug, and transduced cells were fixed and stained for β-Gal activity at 48 h following transduction. ZEBOV-GP-mediated transduction of cells transfected with an irrelevant RNAi is about 4-fold higher than that observed in the presence of Axl RNAi. Each of these transduction values was set to 100% (No Drug), and we assessed the effect of macropinocytosis inhibitors on ZEBOV-GP pseudovirion transduction in the presence or absence of Axl expression relative to these controls. (C) Macropinocytosis inhibitors have no effect on FIV-VSV-G transduction in the presence or absence of Axl in SNB19 cells. Studies were performed as described for panel B, but FIV-VSV-G (MOI, 0.005) was transduced. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05; **, P < 0.001.