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. 2016 Nov 4;7:13287. doi: 10.1038/ncomms13287

Figure 2. GAS5 is essential for hESC self-renewal.

Figure 2

(a) A scheme of lentiviral vectors constructed. The lower panel shows the transefected cell colonies obtained after puromycin selection in fluorescence microscopy (n=3). Lenti-NC represents control lentivirus, Lenti-GAS5 represents GAS5-overexpressing lentivirus and Lenti-shGAS5 represents short hairpin knockdown lentivirus. Scale bar, 500 μm. (b) Colony formation assay of GAS5 overexpression or knockdown hESCs. The scheme of assay is shown above, and the quantitation of colonies is shown in the lower right panel. **P<0.01, t-test, n=3. (c) qPCR and western blot analysis of puipotency genes in different lentivirus transfected cells. **P<0.01, one-way analysis of variance, n=3. (d,e) The cell cycle analysis (n=3) of different stably expressed hESCs and HEK-293T cells (d), the quantification of cell cycle phases in different stably expressed hESCs and HEK-293T cells are shown in e. Ratio of G2 phase cells were compared for significance as indicated. **P<0.01, t-test, n=3. (f) Representative images of EdU analysis (n=3) and flow cytometry quantification (n=3) of different expressed hESCs and HEK-293T cells. (g,h) The mRNA level of positive regulators (g) and negative regulators (h) of cell cycle in different stably expressed hESCs. *P<0.05, **P<0.01, t-test, n=3. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR. See also Supplementary Fig. 2.