Trophoblastic differentiation of shLuc and knockdown NLRP2 (128236 and 130989) H9 embryonic stem cells. (A) Quantitative PCR analysis of the indicated trophoblast markers at days 0 (embryonic stem cell (ESC)), 2, and 7 of differentiation. The relative gene expression was first normalized to that of GAPDH, and then presented as the fold change relative to that in shLuc H9 ESCs. Values are mean ± standard deviation (SD) (n = 3). (B) Immunofluorescence (IF) staining of trophoblast markers at day 7 of differentiation in 128236 knockdown (KD) NLRP2 human embryonic stem cells (hESCs). Scale bars = 50 μm. (C) Quantitative analysis of shLuc and 128236 KD NLRP2 hESCs that exhibited IF staining for both GATA2 and CDX2 at day 7 of differentiation. About 500 to 600 cells were counted for each experiment. Values are mean ± SD (n = 3). *P <0.05. (D) Immunoassays of three placental hormones (human chorionic gonadotropin (hCG), estradiol, and progesterone) at days 0, 2, 4, 6, 8, 10, and 14 of differentiation.