Figure 4.

Overexpression or knockdown of Dax1 results in alteration of endogenous Oct4. A, For overexpression, mES cells were transiently transfected in a six-well plate with 3 μg empty vector or pcDNA3 Dax1, and 48 h later cells were harvested and RNA was isolated. For knockdown, mES cells were transfected in a 10-cm plate with 10 μg of a vector containing shRNA against Dax1 or scrambled control, and a GFP expression cassette. Cells were harvested 24 h after transfection, and GFP-positive cells were sorted and then RNA isolated. For both, cDNA synthesis and QPCR were carried out as described in Materials and Methods. Oct4 values were normalized to glyceraldehyde-3-phosphate dehydrogenase and data presented as fold over empty vector control. *, P < 0.005. B, mES cells were transfected in 10-cm dishes with 10 μg empty vector or pcDNA3 Dax1, and 48 h later cells were harvested and protein was isolated. Western blot analysis was performed as described in Materials and Methods with anti-Dax1, anti-Oct4, and anti-β-actin antibodies. C, mES cells were transfected in a 10-cm plate with 10 μg of a vector containing shRNA against Dax1 or scrambled control, and a GFP expression cassette. Cells were harvested 24 h after transfection and GFP-positive cells were sorted after which cells were harvested and protein was isolated. Western blot analysis was performed as described in Materials and Methods with anti-Dax1, anti-Oct4, and anti-β-actin antibodies. IB, Immunoblotting; shDax1, short hairpin Dax1.