Effects of autocrine TNF-α inhibition in combination with NL on CML SPCs survival and proliferation. (A) CML CD34⁺ cells (n = 3) were either left UT or treated with TNF-α inh (3 µM), NL (5 μM), or their combination for 72 hours before drug washout and plating in methylcellulose progenitor assays. CFC frequency based on their morphology (erythroid-burst-forming unit [BFU-E] and erythroid-colony-forming unit [CFU-E] vs granulocyte/macrophage-colony forming unit [CFU-GM]) was recorded after 12 days of culture. (B) CML CD34⁺ cells (n = 5) were cultured as in panel A for 72 hours, and the percentage of apoptotic cells was measured by annexin staining. (C) CML CD34⁺ cells (n = 4) were CFSE stained and then cultured as in panel A for 72 hours. Percentage of apoptotic cells within the undivided (CFSE^max) population was measured as explained in Figure 1H. (D) Sorted CML, BCR-ABL⁺ (by fluorescence in situ hybridization), CD34⁺ CD38⁻ cells (n = 2) were cultured as in panel A for 72 hours. Percentage of apoptotic cells was measured by annexin staining. (E) Representative flow cytometry plot of CFSE and annexin double-staining showing levels of apoptosis within the CFSE^max population in each treatment group. (F) CML CD34⁺ cells (n = 4) were treated for 72 hours, as in panel A, and the percentage of starting CD34⁺ cells recovered within each division in each treatment group was calculated by recording the number of viable cells seeded initially in each culture and their number after different treatment conditions, using levels of CFSE fluorescence to measure the percentage of cells within each division, as explained elsewhere.¹⁷  All data from independent experiments are presented as mean ± standard error of the mean. *P < .05; †P < .01; ‡P < .001.