Figure 3.

Effects of transduction with lentiviruses expressing HOBIT shRNA on total cell expansion. (A) HOBIT shRNA strongly reduces HOBIT expression. Lentiviral vectors expressing HOBIT shRNA were co-transfected with a HOBIT expression construct into HEK293T cells. After 96 h, cells were either used for RNA isolation or lysed in Laemmli sample buffer. The RNA was used for realtime RT-PCR analysis (upper part). Results are derived from two experiments performed in quadruplicates and shown as mean ± SD. The proteins in the lysed samples were separated by SDS-PAGE, Western blotted, and probed with anti-HOBIT antibodies (lower part). As internal control hypoxanthine-guanine phosphoribosyltransferase was detected by respective antibodies. Two experiments with comparable results were performed. (B,C) HOBIT shRNA reduces expansion of cells at day 21. Cord blood CD34⁺ cells were cultured for 5 days, then cells were transduced with lentiviruses expressing either shHOBIT or a scrambled control shRNA (shControl) or were mock-treated. Transduction efficiency was measured 3 days later by flow cytometry scoring GFP-positive cells. Cells were further cultured and differentiated until day 21. Then flow cytometry was performed to evaluate expansion of transduced GFP-expressing cells. Exemplary dot plots for cells transduced with lentiviruses expressing shHOBIT or shControl or mock-transduced controls are shown in (B). The numbers of the transduced GFP⁺ cells and the non-tranduced GFP⁻ cells in individual cultures was calculated from the measured cell number and the respective percentages determined by flow cytometry and are shown in (C). The values were normalized to the number of transduced or non-tranduced cells measured at day 8 to establish the expansion rates. Results are calculated from four experiments performed in triplicates and are displayed as mean ± SEM (*p < 0.05, ***p < 0.001).