Figure 2.

Effects of KD of Klf5-target genes in ESCs. (a) ESCs stably transfected with shRNA plasmids were grown for 7 days at clonal density (50-100 cells/cm²) and then stained for alkaline phosphatase (AP) activity. The results of shGFP-transfected cells (negative control) and shHck-transfected cells are shown as examples. Scale bars, 1 cm. (b) Histogram showing the percentages of undifferentiated (blue) and differentiated (red) colonies observed by AP staining after knockdown of Klf5 response genes. shKlf5-transfected cells are the positive control, and shGFP-transfected cells are the negative control. Bars represent SD of triplicates. *P < 0.01. (c) qPCR analysis of stemness markers after KD of Klf5 target genes that induced a significant decrease in AP-positive colonies. Efemp1 KD cells were used as negative controls. The data are represented as fold changes relative to shGFP-transfected cells. Error bars represent SD of triplicates. Differences are significant with P < 0.01.