Fig 2: TWEAK treatment of human PC-3 and DU145 prostate cancer cells increases invasion and activates the non-canonical NF-?B pathwayPC-3 A. and DU145 B. cells were either left untreated or treated with 200 ng/ml of TWEAK for 24 hr prior to harvesting and plating 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. The invasion assay was repeated one more time using one chamber per condition with similar results. C. PC-3 and DU145 cells were either left untreated or treated with TWEAK for 24 hr. Cells were harvested and NF-?B2 (p100/52), I?Ba and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice.

Fig 3: Epo elevation in AMPKa1-KO MEFs is required for tumorigenesisA. Plasma concentrations of Epo in nude mice inoculated with WT or AMPKa1-KO MEFs. n=10-13 per group; *P<0.01 versus WT. B. Epo upregulation in inoculated AMPKa1-KO MEFs in nude mice. Implanted MEF tissues were collected from nude mice euthanized by carbon dioxide inhalation at 6 weeks after MEFs implantation. Representative images show Epo staining (top). Quantification of anti-Epo staining (bottom). n=5 per group; *P<0.01 versus WT. C. Tumor growth rate of AMPKa1-KO MEFs in nude mice injected intraperitoneally (IP) with an antibody specific to Epo or IgG. n=8–10 per group, †P<0.01 versus WT/IgG; *P<0.01 versus a1-KO/IgG. D. Representative images showing staining with antibodies specific to CD-31 or smooth muscle (SM)-a-actin (left). Scale bar =50 µm. Quantification of anti-CD31or SM-a-actin staining (right). Implanted AMPKa1-KO MEFs were collected from nude mice euthanized by carbon dioxide inhalation at the end of experiment (8 weeks after MEFs implantation). n=5–6 per group; *P<0.01 versus IgG. E. Mechanisms of AMPKa1deletion-stimulated anchorage-independent growth, neovascularization, and consequent tumorigenesis. AMPKa1 deletion in fibroblast activates NIK, which phosphorylates and activates I?Ka, then enhance p100 phosphorylation recruiting E3 ubiquitin ligase ß-TrCP, which facilitates p100 processing to p52. Upregulated p52 controls CDK2 and Epo expression. Epo-mediated neovascularization cooperating with CDK2-mediated anchorage-independent growth contributes to tumorigenesis in vivo.

Fig 4: Elevated p52 is responsible for Epo inductionA. Upregulated Epo in AMPKa1-KO MEFs. Epo protein level was assessed by Western blot analysis (top). Quantification of Western blot data (bottom). n=5, *P<0.05 versus WT. B. Epo was elevated in culture medium from AMPKa1-KO MEFs. Epo was concentrated from culture medium of MEFs with different genetic background and detected by ELISA. n=6, *P<0.01 versus WT. C. Upregulation of Epo transcription in AMPKa1-KO MEFs. Epo mRNA levels were measured by quantitative RT-PCR in WT, AMPKa1-KO, or AMPKa2-KO MEFs. n=5, *P<0.01 versus WT. D. Epo upregulation in AMPKa1-KO MEFs is mediated by p52. Three kinds of MEFs were transfected with either control siRNA or p52 siRNA (100 nmol/L) for 72 hours. Representative blots from three independent experiments are shown. E. Chromatin immunoprecipitation (ChIP) analysis of the Epo gene. MEF chromatin from WT, AMPKa1-KO, and AMPKa2-KO mice was immunoprecipitated with anti-p52 or rabbit IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for the mouse Epo promoter. This result is representative of four independent experiments. n=4, *P<0.05 versus WT.

Fig 5: 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.