Fig 2: NF-?B signaling contributes to 2'3'-cGAMP/BV6/zVAD.fmk-induced cell death.A Western blot analysis of phosphorylated and total p65, I?Ba, NIK, p100, p52, and TBK1 in the indicated cell lines treated with 4 µg/ml 2'3'-cGAMP and/or 5 µM BV6 for 15 h in the presence or absence of 20 µM zVAD.fmk. GAPDH or ß-Actin serves as loading controls. Representative blots of at least two different independent experiments are shown. B Western blot analysis of p65 in the indicated PC cell lines after 48 h of transfection with non-silencing RNA (ctrl) or siRNA targeting p65. ß-Actin serves as loading control. Representative Western blots of at least two different independent experiments are shown. C AsPc-1 and BxPc-3 cells were transfected with control siRNA (siCtrl) or two independent siRNAs targeting p65 and were treated with 4 µg/ml 2'3'-cGAMP and 5 µM BV6 for 48 h in the presence of 20 µM zVAD.fmk. The amount of cell death was calculated by quantifying PI uptake determined with the ImageXpress Micro XLS system. Data are presented as percentage of PI-positive cells, and mean and SD of three independent experiments performed in triplicate are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Effects of TRAF6 silencing on the expression levels of p-p65 and p-p100 protein following the addition of patient stromal cells. (A) The TRAF6 siRNA-transfected U266 and RPMI8226 myeloma cells were co-cultured with patient stromal cells for 24 h prior to immunoblotting with anti-p-p65 and anti-p-p100. (B) The two TRAF6 siRNA-transfected cell lines exhibited significantly decreased expression levels of p-p65 and p-p100, compared with non-transfected cells (*P<0.05). (C) The U266 and RPMI8226 cells were co-cultured with patient stromal cells for 24 h prior to immunoblotting with anti-p65, anti-p100 and anti-p52. siRNA, small interfering RNA; TRAF6, tumor necrosis factor receptor-associated factor 6; p52, tumor protein 52; p-p65, phospho-p65; P-100, phospho-100; NC, negative control.

Fig 4: NF-?B2 p100 depletion attenuates TWEAK-stimulated DU145 cell invasionA. DU145 cells were either left untreated (NT, no treatment) or transiently transfected with control (CTL) siRNA or p100 siRNA #4, treated with 200 ng/ml TWEAK (TWK), and harvested 24 hr post-transfection. Lysates were prepared and NF-?B2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice. B. DU145 cells were either left untreated or transiently transfected with CTL siRNA or p100 siRNA #4, treated with 200 ng/ml TWEAK, harvested 24 hr post-transfection and plated in Matrigel invasion chambers. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test (ns = not significant; p > 0.05). The invasion assay was done twice and the results of one representative experiment are shown here.

Fig 5: NF-?B2/p52 elevation in AMPKa1-KO MEFs occurs via ß-TrCP-mediated p100 processingA. AMPKa1 deletion upregulates both p52 and RelB protein levels in both cytoplasm and nucleus of MEFs. p52, RelB, GAPDH, and histone H3 proteins in subcellular fractions of WT, AMPKa1-KO, and AMPKa2-KO MEFs were analyzed by Western blot (top). Quantification of the p52 and RelB data (bottom). n=4, *P<0.05 versus WT/cytoplasm; †P<0.05 versus WT/nucleus. B. Levels of p100 and p52 proteins in WT, AMPKa1-KO, and AMPKa2-KO MEFs. p100 and p52 protein levels were assessed by Western blot analysis (top). This blot is representative of five blots from five independent experiments. Quantification of western blot data (bottom). n=5, *P<0.01 versus WT. C. Increased phosphorylation of p100 at Ser-866/870 (pp100-S866/870) and I?B kinase-a at Ser176 (pI?Ka) in AMPKa1-KO MEFs. pp100-S866/870, pI?Ka, and I?Ka protein levels were assessed by Western blot (top). This blot is representative of five independent experiments. Quantification of Western blot data (bottom). n=5, *P<0.01 versus WT. D. Elevated phosphorylation of NIK at T559 (pNIK-T559) in AMPKa1-KO MEFs. pNIK-T559 and NIK protein levels were assessed by Western blot (top). Quantification of Western blot data (bottom). n=5, *P<0.01 versus WT. E. Enhanced interaction of p100 with pI?Ka in AMPKa1-KO MEFs. pI?Ka-S176 was detected by immunoblotting after immunoprecipitation (IP) with anti-p100 (top). Quantification of Western blot data (bottom). n=5, *P<0.01 versus WT. F. Augmented association of ß-TrCP with pp100 in AMPKa1-KO MEFs. pp100 was detected by immunoblotting after immunoprecipitation (IP) with anti-ß-TrCP (top). Quantification of co-immunoprecipitation data (bottom). n=4, *P<0.01 versus WT. G. p52 elevation in AMPKa1-KO MEFs is mediated by ß-TrCP. MEFs were transfected with either control siRNA or ß-TrCP siRNA (100 nmol/L) for 72 hours. Representative blots from three independent experiments are shown (top). Quantification of Western blot data (bottom). n=3, *P<0.05 versus WT/control siRNA; †P<0.05 versus a1-KO/control siRNA.