Nucleofection of hES cells with DNA, shRNA vectors, or siRNAs alters gene expression. (A): Flow cytometric analysis of GFP and SSEA-4 expression in H1 and H9 hES cells 4 days after transfection with the GFP vector (middle and right) or no DNA (left). (B): BF (top) and GFP expression (bottom) of hES cells 4 days after co-transfection of the GFP vector and either β2M siRNA or Oct4 siRNA. GFP expression was used to identify transfected cells. (C): BF (top) and SSEA-4 (bottom) immunostaining of β2M KD and Oct4 KD hES cells. (D): AP staining of β2M KD and Oct4 KD hES cells. (E): Reverse transcription-polymerase chain reaction analysis of hES cells transfected with either shRNA vectors or siRNAs. β-Actin was used as a template loading control. Abbreviations: AP, alkaline phosphatase; BF, bright-field; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; hES, human embryonic stem; KD, knockdown.