Dissecting Early Differentially Expressed Genes in a Mixture of Differentiating Embryonic Stem Cells
Figure 2
Depletion of the candidate self-renewal factor Smarcad1 by RNAi.
Three shRNA constructs were used to target different regions of respective transcripts. (A) Two days after puromycin selection, the colony morphology of typical undifferentiated ES cells with positive alkaline phosphatase (AP) staining (red) was maintained in two control experiments (Empty and Luci) and three Pias2 knockdown experiments. In contrast, flattened fibroblast-like cells were formed in each Smarcad1 knockdown experiment, and AP staining in Smarcad1 depleted cells was reduced. (B) Quantitative real-time PCR analysis of gene expression in four-day knockdown ES cells. The levels of the transcripts were normalized against the control experiment of empty vector transfection. Data are presented as the mean±SEM, which was derived from three independent experiments.