TCFAP2C, SMARCA4, and EOMES are required for TS cell self-renewal. (A) Tcfap2c, Smarca4, and Eomes siRNAs used to deplete Tcfap2c, Smarca4, and Eomes mRNA in TS cells. Control siRNA, Tcfap2c siRNA, Smarca4 siRNA, Eomes siRNA, Tcfap2c/Smarca4/Eomes siRNA, Tcfap2c/Smarca4 siRNA, Tcfap2c/Eomes siRNA, or Smarca4/Eomes siRNA were nucleofected into TS cells every 2 d for 6 d. (B) Normal colony morphology was lost in TS cells nucleofected with Tcfap2c siRNA, Smarca4 siRNA, Eomes siRNA, Tcfap2c/Smarca4/Eomes siRNA, Tcfap2c/Smarca4 siRNA, Tcfap2c/Eomes siRNA, or Smarca4/Eomes siRNA compared with TS cells nucleofected with control siRNA. (C) Immunofluorescence analysis of TS cells conucleofected with Tcfap2c/Smarca4/Eomes siRNA. TCFAP2C, SMARCA4, and EOMES expression was down-regulated in TS cells nucleofected with Tcfap2c/Smarca4/Eomes siRNA compared with TS cells nucleofected with control siRNA. Cytoplasm was stained with an antibody specific to Cytokeratin (Cytok) and nuclei were stained with Hoechst 33258. Scale bars, 100 μM. (D) qRT-PCR expression analysis of Tcfap2c, Smarca4, and Eomes siRNA TS cells with control TS cells. Expression of TS cell enriched genes were down-regulated following nucleofection of Tcfap2c, Smarca4, and Eomes siRNA. (E) Heat map summary of qRT-PCR results.
