A) pre-iPSCs derived from Nanog-GFP reporter MEFs by retroviral expression of Oct4, Sox2, Klf4, and cMyc were transfected once with the indicated siRNAs. Reprogramming efficiency was determined by FACS analysis of GFP-positive cells on day 14 post siRNA transfection (i) or by counting the number of GFP-positive colonies on day 15 (ii). Note: colony numbers presented for Cbx3 and 3XHMT single knockdowns are the same as in Fig 6Diii, tetO-Nanog(−)/dox(−) condition). Repeated transfection of the siRNAs did not result in an increase in the number of GFP-positive cells.
B)
Nanog-GFP-positive ESC-like colonies obtained upon Cbx3 knockdown in pre-iPSCs were expanded and two lines (iPSCs-Cbx3 #1 and #2) analyzed by qRT-PCR for transcript levels of the pluripotency genes Esrrb and Nanog. For comparison, ESCs, MEFs, and the starting pre-iPSCs, which were set to 1, are included in the analysis. Data presented is the mean of three technical replicates from one experiment.