GHFT1 stable cell lines

GHFT1 cells (Dr Pamela Mellon, University of San Diego, La Jolla, CA) were maintained at 37°C/5% CO₂ in Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (Hyclone, Logan, UT) and 100 units/ml penicillin-streptomycin (Invitrogen). All cultured cell lines employed in this study were authenticated by RNA-Seq analysis performed in the lab (Figure 8—source data 1). This authentication was done based on the positive expression of Pou1f1 and absence of expression of Prolactin, Growth hormome and Thyroid-Stimulating Hormone. Prop1 cDNA was cloned into pEF1α-FLBIO-puro (Kim et al., 2009) to be N-terminally tagged by standard cloning using the BamHI restriction site. The BirA expression vector used was pEF1α-BirAV5-neo (Kim et al., 2009). Both plasmids were a gift from Alan B. Cantor (Boston Children’s Hospital, Boston, MA). Cells were plated onto 24-well plastic plates (Fisher Scientific, Fair Lawn, NJ) at a density of 3.5 × 10⁴ cells/well. Prop1 and BirA expressing vectors were transfected into cultured cells using the FuGENE 6 (Promega) at a 3:2 ratio according to the manufacturer’s protocol. To make stable cell lines, transfected cells were incubated with 1.4 mg/ml G-418 solution (Roche) and 5 µg/ml Puromycin (Sigma) for selection of BirA and Prop1-Tag, respectively.