Figure 6. Rescue of Msi2 knockdown using an epitope-tagged overexpression construct.

Cells engineered to overexpress Flag-tagged Msi2 isoform 1 or Flag-tagged Msi2 isoform 2, i-Msi2.1-D3 and i-Msi2.2-D3 respectively, were plated at 10,500 cells/cm² in the presence or absence of 2 µg/ml Dox. Cells were infected with lentiviral constructs that express Msi2 shRNA #1 for 48 hours. One day later, the cells were subcultured at 600 cells/cm² in the continued presence or absence of 2 µg/ml Dox and the remaining cells harvested for protein extracts. Western blot analysis was used to monitor the levels of Msi2 in i-Msi2.1-D3 (A) or i-Msi2.2-D3 (B) stably infected with shRNA #1 lentivirus in the presence or absence of 2 µg/ml Dox. D3 ESC infected with the lentiviral vector that expresses the scrambled shRNA was used as a control. (C) Effects of inducing Flag-tagged Msi2 isoform 1 (left) or Flag-tagged Msi2 isoform 2 (right) on the cloning efficiency of D3 ESC following Msi2 knockdown. Six days following subculture, two observers unaware of sample designation scored colonies as ESC, Mixed or differentiated (Diff). The error bars are standard deviation between the average percentages as scored by the two observers. This experiment was repeated twice and similar results were obtained.