Autocrine TNF-α production in CML SPCs is BCR-ABL kinase-independent, induces NFκB/p65 activity, and promotes their survival. (A) TNF-α blood plasma levels were measured by enzyme-linked immunosorbent assay in CP (n = 24) and accelerated phase (AP) (n = 3) CML patients. Levels are expressed as picograms per milliliter. The range of TNF-α blood plasma levels in normal controls (n = 8) is shown in the shaded area. (B) TNF-α mRNA expression levels were measured by qRT-PCR and normalized to the control genes ATP5B, B2M, ENOX2, GUSB, TBP, and TYW1 mRNA expression levels in newly diagnosed CP CML (n = 30) and normal (n = 4) CD34⁺ cells. (C) TNF-α protein expression was measured by intracellular flow cytometry in CML (n = 6) and normal (n = 4) CD34⁺ cells and expressed as a ratio of the mean fluorescence intensity (MFI) of TNF-α antibody-stained cells over the MFI of cells stained with a matched isotype control. (D) CML CD34⁺ cells (n = 4) were either left untreated (UT) or treated with NL (5 µM) for 48 hours, and TNF-α protein expression was measured by intracellular flow cytometry, as explained in panel C. TNF-α expression levels in the NL-treated cells were expressed as a percentage of UT cells. (E) CML CD34⁺ cells (n = 3) were either left UT or treated with TNF-α inhibitor (TNF-α inh) (3 µM) or TNF-α inh (3 μM) + TNF-α (1 ng/mL). Levels of p-NFκB/p65^Ser536 were measured by intracellular flow cytometry at 24 hours, as described in panel C, and expressed as a percentage of UT. (F) IAP2 gene expression levels were measured at 24 hours by qRT-PCR after treatment, as in panel E. Differences in gene expression levels after treatment were calculated using the 2^−ΔΔCt method, after normalization within each sample of candidate gene expression levels against GAPDH and TBP expression levels. Relative quantification (RQ) of IAP2 mRNA expression after TNF-α inh treatment was then plotted as log₂ of the 2^−ΔΔCt values (with the UT cells having a value of 0 in the graph being the calibrator). (G) CML CD34⁺ cells (n = 5) were either left UT or treated with TNF-α inh (3 µM) or TNF-α inh (3 μM) + TNF-α (1 ng/mL) for 72 hours. Percentage of apoptotic cells was measured by annexin staining. (H) CML CD34⁺ cells (n = 3) were CFSE stained and then cultured as in panel G for 72 hours. Percentage of apoptotic cells within the undivided (CFSE^max) population was measured by gating on the population double-positive for maximal CFSE expression and annexin staining. All data from independent experiments are presented as mean ± standard error of the mean. *P < .05; †P < .01; ‡P < .001; ns, not significant.