Stable cell lines expressing fluorescent proteins

AU565 (ATCC CRL 2351) and MDAMB157 (ATCC HTB 24) cells were grown in DMEM supplemented with 10% FBS, HCC1143 (ATCC CRL 2321) cells were grown in RPMI supplemented with 10% FBS, and 21MT1 (generous gift from Kornelia Polyak) cells were grown in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml rhEGF, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 µg/ml insulin. Cells were genetically engineered using plasmids available from Addgene. The coding fragment for clover-HDHB (Addgene plasmid 136461)^{23 (link)} was cloned in frame into a transposase expression plasmid modified to also express a nuclear localized mCherry and stable cell lines were created as previously described⁴⁴ . In brief, the clover-HDHB-NLS-mCherry expression plasmid was co-transfected 24–48 h with the pSB100X transposase plasmid (Addgene plasmid 34879) at a ratio of 4:1 using Lipofectamine 3000 (AU565, HCC1143, 21MT1) or LTX (MDAMB157) and selected for 3–7 days with 0.5–2 µg/ml puromycin. To ensure uniform fluorescence across the transfected population, HCC1143 and 21MT1 cells were sorted at OHSU’s Flow Cytometry Core and cells with a medium intensity clover-HDHB signal and a high intensity NLS-mCherry signal were selected for drug dose response experiments. In all cases, cells were validated by STR profiling (LabCorp) and tested negative for mycoplasma.