(A) LOUCY_MEF2C-BFP cells were cultured in triplicate without (dashed lines) or following induction of MEF2C (+dox, solid lines) in media containing 0%, 1%, or 10% fetal calf serum (FCS; mean values ± SD are shown). (B) Percentage of cells expressing the MEF2C-BFP construct in the derivative LOUCY_MEF2C-BFP bulk line. Triplicate experiments grown for 22 days in the presence of 1% (left) or 10% (right) FCS. Each column displays the fraction of MEF2C-BFP expressing cells in blue. (C) LOUCY_MEF2C-BFP cells were grown in medium containing 1% FCS without (–dox, gray line) or with MEF2C-BFP induction (+dox, blue line). Dox was washed out at day 17 (dashed blue line), and cells were further cultured in 1% FCS media without dox. Mean values ± SD from an experiment in triplicate are shown; shown are representative examples of 3 independent experiments performed. (D) Western blot for MEF2C and BCL2 in LOUCY_MEF2C-BFP cells that were grown in 1% or 10% FCS media for 3 days without (–dox) or with MEF2C induction (+dox). Relative band intensities for BCL2 normalized to β-actin are indicated, with the –dox condition ratio set at 1. (E) Western blot of phospho-STAT5 (Tyr694), total STAT5, and BCL2 in LOUCY_MEF2C-BFP cells that were grown in 10% FCS media without (–dox) or with MEF2C induction (+dox) for 24 hours and subsequently incubated without and with 100 ng/mL IL-7 for 15 minutes (top) or 2 days (bottom). Ruxolitinib (2 μM) was added for 1 hour before IL-7 addition and remained present throughout the experiment. β-Actin was used as a loading control. Relative band intensity ratios for phospho-STAT5 (normalized to total STAT5 level) and for BCL2 (normalized to β-actin) are shown as explained in D.