Fig. 6: Functional assessment of TFAP2C actions in trophoblast cell development.

a Representative human placental tissue specimen (12 weeks of gestation) probed for TFAP2C, CDH1, and PLAC8 transcripts using in situ hybridization. 4′,6-diamidino-2-phenylindole (DAPI) labels cell nuclei. Overlay of merged immunofluorescence images: TFAP2C (magenta), DAPI (gray), and either CDH1 (cyan) or PLAC8 (cyan), respectively. Scale bars represent 50 μm (CDH1 image panels) and 100 μm (PLAC8 image panels). b RT-qPCR measurement of TFAP2C normalized to POLR2A in stem (red) and extravillous trophoblast (EVT) cells (blue). Data were analyzed by unpaired t-test and are presented as mean values ± standard deviation (SD; ns = not significant p = 0.8374; n = 4 biologically independent replicates per group). c TFAP2C (50 kilodaltons (kDa)) and GAPDH (37 kDa) proteins in stem and EVT cells assessed by western blot analysis. d Log₂ fold-change values of normalized read counts of TFAP2C from RNA-Seq in stem state and days 3, 6, and 8 of EVT cell differentiation compared to the stem state. e RT-qPCR measurement of TFAP2C normalized to POLR2A in stem state cells transduced with lentivirus containing a control shRNA (shC; blue) or a TFAP2C-specific shRNA (gray). Data were analyzed by unpaired t-test and are presented as mean values ± standard deviation (SD; shTFAP2C; n = 4 biologically independent replicates per group; **p = 0.0029). f TFAP2C (50 kDa) and GAPDH (37 kDa) proteins in stem state cells transduced with shC or shTFAP2C and assessed by western blot. g Phase contrast images of stem state cells transduced with shC or shTFAP2C. Scale bar represents 250 μm. h Heat map based on scaled, normalized counts of selected differentially expressed transcripts generated from RNA-Seq in shC (control, Ctrl; orange) or shTFAP2C (knockdown, KD; blue) transduced cells using a red-blue diverging scale. Transcripts are clustered into four groups including cell cycle (purple), EVT-specific (pink), stem state-specific (green), or other (blue). *Depict genes identified as direct targets of TFAP2C based on ChIP-Seq analysis in stem state cells. Graphs in panels (b) and (e) depict mean ± standard deviation. Source data are provided as a Source Data file.