RET activation by GDNF/GFRα1 sustains an NF-κB/p53/BCL2 anti-apoptotic program in HSPCs during in vitro culture. (A) Bar graphs depict median intensity of signal from histograms below showing the profiles of key protein changes in CD34⁺ cells at day 0 (blue), day 3 control (Ctrl; orange), day 3 GDNF/GFRα1 (green), day 7 Ctrl (red), and day 7 GDNF/GFRα1 (purple; a.u., arbitrary units). (B) z-normalized heatmap of data in panel A, illustrating differences in CD34⁺CD38^– cells at input, and CD34⁺ cells at day 3 and day 7 culture with or without GDNF/GFRα1 treatment assayed by mass cytometry, supervised by treatment condition. (C) Fold change RNA expression of key NF-κB target genes in GDNF/GFRα1–treated CD34⁺CD38^– cells compared with Ctrls at days 1, 3, and 7. Gene names are noted under bar labels. A Student t test was used to calculate significant differences. (D) Fold change RNA expression of key genes altered at the protein level in GDNF/GFRα1–treated CD34⁺CD38^– cells compared with Ctrls at days 1, 3, and 7. Gene names are noted under bar labels. A Student t test was used to calculate significant differences. (E) Illustrated pathway identified through kinome, mass cytometry, and RNA changes, defining activating (green) and inhibiting (red) phosphorylations, protein levels or RNA levels, and proposed modes of action. For panels C-D, *P < .05, **P < .005; N = 3 per condition and day tested.