Knockdown of HPRT provides hematopoietic cells with resistance to 6TG. Fl5.12 cells were transduced with control (Ctrl) and experimental constructs (490-494) with sequences coding for shRNA against HPRT. The cells were then selected in puromycin for 5 days. (A) Whole cell lysates were subject to Western blot analysis using antibodies against HPRT (ab10479; Abcam, Cambridge, MA) and actin (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA). (B) Proliferating cells were cultured at an initial concentration of 5 × 10⁴ cells/mL in the presence of 6TG at 10, 20, 40, 60, and 100 nM or no drug (UT). The concentration of live cells after 96 hours is depicted as a percentage of Ctrl UT live cells (P < .001; ANOVA). (C) Transduced FL5.12 cells were plated at 10⁵ cells/mL in the presence of 5 nM and 10 nM 6TG or no drug. Cells were replated at 10⁵ cells/mL in fresh media with or without 6TG every 48 hours. Population doublings are plotted against time (P < .001 for 491 [6TG 5 nM] compared with Ctrl [6TG 5 nM]: ANOVA). Population doubling was calculated by taking the log₂ of the cumulative expansion. Cumulative expansion was calculated by multiplying the interval expansion (measured cell concentration/seeded concentration) by the cumulative expansion. (D) Ctrl- and 491-transduced cells were cultured with cisplatinum or no drug. Cells were counted after 48 hours. The number of population doublings is depicted as a percentage of untreated Ctrl-transduced cells' population doublings. ***P < .001.