NPR-A knockdown decreases ES pluripotency genes and upregulates differentiation genes. (a) Real-time PCR of mRNA levels of pluripotency genes (Oct4, Nanog and Sox2) after treatment of ES cells with control siRNA or NPR-A siRNA. (b) RT-PCR analysis of ES cells treated as described in panel a, showing expression of stem cell marker genes. GAPDH was used as the internal control. (c) Real-time PCR of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes, GATA-4 (GATA-binding protein 4), GATA-6 (GATA-binding protein 6), AFP (α-fetoprotein), Brachyury, nestin, Cdx2 (caudal-type homeobox 2), Hand1 (heart and neural crest derivatives expressed transcript 1) and Eomes (eomesodermin homolog). (d) RT-PCR analysis of ES cells treated as described in panel a, showing mRNA levels of the early differentiation genes. GAPDH was used as the internal control. Data represent mean±S.D. (n=3); *P<0.05 (two-tailed t-test)