FIG 7.

Ouabain increases AcMNPV transduction of mammalian cells by decreasing early endosome (EE) pH. (A) Sf9 and HeLa cells were treated with ouabain and infected/transduced by Ac-BPegfp at an MOI of 0.2 (Sf9 cells) or 5 (HeLa cells) TCID₅₀ units/cell. Cells were imaged at 16 h p.i. (Sf9 cells) or 24 h p.t. (HeLa cells). (B) FCM analysis of the effect of ouabain on virus transduction/infection as described in the legend to panel A. (C) Cells were treated with/without 0.8 μM ouabain, transduced by recombinant viruses containing mutated GP64 at an MOI of 5 TCID₅₀ units/cell, and analyzed by FCM at 24 h p.t. The results were normalized to those of WT virus treated without ouabain. (D and E) Ouabain decreased the EE pH. Sf9 (D) and HeLa (E) cells were transfected with plasmids expressing Rab5^mCherry or Rab7^mCherry. Cells were incubated with 0.8 μM ouabain and dextrans conjugated to FITC or Alexa Fluor 647. Cells were imaged via fluorescence microscopy, and then the relative endosomal pHs were calculated by ratiometric fluorescence measurements. The results are mean values ± SD (n = 4 independent experiments). (F) Effects of ouabain on virus transduction of different mammalian cells. Each cell line was treated with ouabain and transduced by Ac-BPegfp at an MOI of 5 TCID₅₀ units/cell, followed by analysis with FCM at 24 h p.t. For panels B, C, and F, all results are mean values ± SD (n = 3 experimental replicates). Statistical analysis was performed using the unpaired t test: *, P < 0.05; ***, P < 0.001; ns, not significant.