Figure 3.

Tnf signal inactivation sensitizes LC to NF-κB inhibitor treatment but protects healthy HSPC from such treatment. (A and B) Tnf stimulation of NF-κB activity in both LC and HSPC as shown by p65 (NF-κB1) nuclear localization (values in parenthesis are mean similarity dilate peak, sample size = 5,000 cells) as determined by ImageStreamX in A or by p65 phosphorylation in B. Bars, 10 µM. (C) HSPC and LC stimulated with individual cytokines show different patterns of p65 phosphorylation. (D and E) HSPC and LC were treated in parallel with indicated doses of BAY11-7085 (BAY) for CFU assay in D or analyzed for cell death in E. (F) HSPC and Tnfr^−/− HSPC were treated with increasing doses of BAY and plated for CFU assay. (G) HSPC and Tnfr^−/− HSPC were transduced with IκBαSR-GFP. GFP⁺ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC was normalized to vector-only-transduced corresponding genotype control. (H) LC and Tnfr^−/− LC were treated with increasing doses of BAY and plated for CFU assay. (I) LC and Tnfr^−/− LC were transduced with IκBαSR-GFP. GFP⁺ cells were sorted and plated for CFU assay. The number of CFUs in each genotype of HSPC or LC was normalized to vector-only-transduced corresponding genotype control. (J) LCs were co-treated with BAY and TNF. CFUs were measured 1 wk after treatment. (K) Experiment model. All values shown are mean ± SD normalized to vehicle-treated control, performed on three independent trials. * (P < 0.05) and ** (P < 0.01) indicate significant reduction compared with corresponding vehicle groups or vector-only groups as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 and ## indicates P < 0.01 significant difference when compared with indicated conditions.