Classical NF-κB signaling is dispensable for d.a.MKK6-induced UCM. (A) U937Te cells were infected with either empty pBMN-IRES-GFP vector or pBMN-d.a.MKK6-IRES-GFP. GFP⁺ cells were analyzed by FACS for the expression of CD86, CD83, and CD40. Bars represent mean percentages ± SD of 3 independent experiments (*P < .05, **P < .01). (B) U937 cells were cotransduced with TA-mCD8α and HR-NGFR or d.a.MKK6-NGFR, and optimal inducible clones were generated. FACS diagrams show NGFR versus mCD8α expression of a d.a.MKK6-inducible U937 clone grown with or without DOX for 48 hours. Representative Western blot analysis of cell lysates from d.a.MKK6- or control vector–inducible U937 cells treated with DOX (1 μg/mL) for the indicated time points. MKK6, phosphorylated or total p38, and phosphorylated or total JNK were detected. One representative of 4 independent experiments is shown. (C) Representative Western blot analysis of nuclear and cytosolic extracts from a d.a.MKK6-inducible U937 clone transduced with either pBMN-IRES-GFP, pBMN-IκB-SR-IRES-GFP, or pBMN-p100ΔN-IRES-GFP grown with or without 2 μg/mL DOX (24 hours). GFP⁺ cells were FACS sorted prior to preparation. Extracts were probed with anti-p65, anti-RelB, antiactin (cytosolic control), and anti-PCNA (nuclear control) antibodies. (D) Representative FACS blots of a U937T NF-κB–GFP reporter cell line coinfected with empty vector controls or d.a.MKK6-NGFR together with pBMN-IRES-mCD8α or pBMN-IκB-SR-IRES-mCD8α. NGFR⁺ mCD8α⁺ cells were analyzed for CD86 versus GFP. Histogram overlay shows GFP induction (NF-κB activity) in response to TNFα stimulation (2.5 ng/mL, 48 hours) of control transduced cells (bold black line) compared with cells grown in the absence of TNFα (full gray line) or cells transduced with d.a.MKK6 (thin black line). One representative of 3 independent experiments is shown.