Figure S5.

Gating strategy for FACS of ESCs or pESCs overexpressing tagged proteins. (A, C, and D) Schematic representation of the overexpression strategy. (B and E) Representative example of the FACS sorting of pESCs overexpressing LRP6-eGFP (B), ESCs overexpressing N-cadherin–eGFP, or pESCs overexpressing E-cadherin–mCherry (E). Gates used were SSC-A versus FSC-A for cells, SSC-A versus SSC-W for single cells, FSC-A, DAPI⁻ for alive cells, followed by sorting by eGFP (FITC) or mCherry (PE-CF549). Control populations were used to set DAPI⁺/DAPI⁻, eGFP⁺/eGFP⁻, and mCherry⁺/mCherry⁻ gates. eGFP⁺ cells were sorted for LRP6-eGFP (pESC) and N-cadherin–eGFP (ESC) conditions. mCherry⁺ cells were sorted for E-cadherin–mCherry condition. CMV, cytomegalovirus promoter; CNTRL, control; Ecad, E-cadherin; mCh, mCherry; Ncad, N-cadherin.