FIG 6.

Genomic RNA delivery to cytoplasm upon 5-NT treatment. Pretreated U-2 OS cells with vehicle control DMSO, 5-NT, or bafilomycin A1 (BafA1) were infected with CHIKV-LR 2006 OPY1 at an MOI of 5 for 1.5 h at 37°C. The cells were washed and permeabilized using 50 μg/ml digitonin for 5 min at RT, followed by 30 min on ice. Separation of the cytoplasmic fraction from the membrane fraction was assessed by Western blot analysis. Subsequently, the total number of GECs was determined in the cytoplasmic and the membrane fraction. (A) Representative Western blot image of the separation. In total, three independent experiments were performed. Shown is GAPDH, which represents the cytoplasmic fraction (C), and EEA1 and Rab5, which represent the membrane fraction (M) from the extracted cells. (B) Total number of viral GECs was assessed by RT-qPCR after Western blot confirmation of successful fractionation. The fold change of cytoplasmic fraction of total gRNA copies is shown. Three independent experiments were performed, each in duplicate. The interpretation of each dot, bar, error bar, and statistic is explained in the legend to Fig. 1.