Fig 8.

Expression of SLC35A1 wild-type (WT) and T128A mutant restores rAAV5 transduction and nuclear import, but not the ∆C Tail mutant in HEK293^SLC35A1 cells. HEK293^SLC35A1-KO cells were transduced with a lentiviral vector that expressed SLC35A1 WT, T128A, and ∆C Tail, as indicated, or untransduced (Mock), followed by the selection of blasticidin (at 10 µg/mL) for a week. The blasticidin-resistant cells were transduced with rAAV5 at an MOI of 20,000. HEK293^Scramble cells were used as a control. (A) rAAV transduction efficiency. At 3 dpt, luciferase activities were measured and normalized to the Scramble (set as 1.0). Data shown are means with an SD from three replicates. P values were determined by using one-way ANOVA for the comparison of the fold changes in the SLC35A1 KO cell groups and the Scramble cell control. (B) rAAV genome distribution. After 12 hpt, nuclear and the cytoplasmic fractions of the rAAV5-transduced were fractionationed, and the vector genomes in each fraction were quantified by qPCR. The percentage of viral genome in each fraction shown are means with an SD of three replicates. P values were determined by using one-way ANOVA for the comparison of the vector genome copies in the nucleus between the SLC35A1 KO cell groups and the Scramble cell control. (C–F) Flow cytometry of lectin staining. The cells were stained with biotinylated SNA (C&D) or MALII (E&F) lectin and FITC-conjugated streptavidin, followed by flow cytometry. The mean fluorescence intensity (MFI) values were calculated, normalized to the WT HEK293 cells as percentages, and shown as means with an SD from at least three replicates. P values were determined by using one-way ANOVA for the comparison of the fold changes in the SLC35A1 KO cell groups and the Scramble cell control.