Mitochondrial DNA-depleted human skin fibroblasts (HSF rho0) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes encoding numerous protein classes in NF-kappaB and STAT3 signaling pathways and by decreased activity of the mitochondrial death pathway compared to the parent rho+ HSF. In contrast the extrinsic TRAIL/TRAIL-Receptor-mediated death pathway remained highly active and exogenous TRAIL induced higher levels of apoptosis in rho0 cells compared to rho+ HSF. Global gene expression analysis using microarray and quantitative RT-PCR demonstrated that expression levels of many growth factors and their adaptor proteins (FGF13 HGF IGFBP4 IGFBP6 IGFL2) cytokines (IL6 IL17B IL18 IL19 IL28B) and cytokine receptors (IL1R1 IL21R IL31RA) were substantially decreased after mitochondrial depletion. Some of these genes were targets of NF-kappaB and STAT3 and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation induced expression of several NF-kappaB/STAT3 target genes including IL1A IL1B IL6 PTGS2/COX2 and MMP12 in rho+ HSF but this response was substantially decreased in rho0 HSF. Suppression of the IKK-NF-kappaB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated rho+ HSF. Inhibitory antibodies against IL6 the main activator of JAK2-STAT3 pathway added into the cell media also increased TRAIL-induced apoptosis in rho+ HSF. However NF-kappaB activation was partially lost in mitochondrial DNA-depleted HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-kappaB targets further suppressing IL6-JAK2-STAT3 that in concert with NF-kappaB regulated protection against TRAIL-induced apoptosis. There are 12 total samples 3 biological replicates each of HSF rho+ and rho0 cells that were not irradiated (control=C) or irradiated (alpha=A). Cells were harvested at 4 hours after treatment.