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. 2015 Dec 21;10(12):e0145280. doi: 10.1371/journal.pone.0145280

Fig 1. Knockdown (KD) of REST in human embryonic stem cells does not result in loss of pluripotency.

Fig 1

A. H9 hESC colonies with an inducible shRNAmir expression system to knockdown REST (REST KD) or a scrambled shRNAmir Non-Target vector as a control (NT) are shown. RFP expression demonstrates near homogeneity of established NT or REST KD lines. B. Immunofluorescence analysis demonstrated reduced REST protein expression in REST KD H9 hESCs. C. Western Blot analysis demonstrated reduced REST protein expression in REST KD H9 and H1 hESCs. D. Expression of pluripotency markers using qPCR. REST levels are statistically significantly knocked down (p<0.0001) in H9 and H1 REST KD hESCs, and there is no change in the expression of OCT4, SOX2 or NANOG, compared to control NT lines. Error bars represent standard error of three independent experiments and asterisks denote a p value < .05 using Student’s t-test analysis. E. Western blot shows no change in protein levels of OCT4, SOX2 or NANOG in REST KD H9 hESCs compared to control NT. F. FACS analysis shows no change in SSEA4 and TRA1-81 levels in REST KD hESCs. G. REST KD and NT hESCs are pluripotent and form teratomas in vivo. Cells from all three germ layers are shown with H&E staining and labeling in black.