Fig 2: NNU219 induces apoptotic signal transduction and modulates the NF-?B signaling pathway in human MM cell lines. (A) The MM cell lines RPMI 8226, ARH77 and MM.1S were incubated with DMSO or NNU219 (10 and 20 nM) for 24 or 48 h, respectively. Cells were fixed with ethanol and stained with propidium iodide, then DNA content was determined by flow cytometry (p < 0.05 for all cell lines). (B) The MM cell lines RPMI 8226, ARH77 and MM.1S were incubated with DMSO or NNU219 (10 and 20 nM) for 24 or 48 h and induction of apoptosis was determined after annexin V-FITC/propidium iodide staining by flow cytometry. Data were presented as mean ± SD of three independent experiments (p < 0.001 for all cell lines). (C–F) RPMI 8226 cells were incubated with NNU219 (10 and 20 nM) for 24 h. After the incubation, cells were lysed and directly subjected to SDS-PAGE, transferred to membranes and blotted with indicated antibodies. GAPDH immunoblotting was included for protein loading control. Blots in the figures were representatives of three independent experiments. (G) MM1.S and RPMI 8226 cell lines were treated with 10 nM of NNU219 for 24 h. The stimulated cells were lysed and 20 µg of protein was processed for ubiquitin immunoblotting. (H) MM1.S and RPMI 8226 cells were treated with DMSO, 10 nM of NNU219 for 24 h and immunoblotted using anti-I?Ba and anti-phospho-I?Ba antibodies. (I) MM1.S and RPMI 8226 cell lines were treated with 10 nM of NNU219 for 24 h. Correlative proteins were immunoprecipitated from 1 mg of MM cell lysate using I?Ba or IgG antibody (rabbit) and coupled to protein A/G agarose beads. The beads were washed by IP buffer and processed by immunoblotting for ubiquitin, p65 or I?Ba. (J) MM1.S and RPMI 8226 cell line was treated with 10 nM of NNU219 for 24 h. Nuclear and cytoplasmic protein fractions were separated from the total lysate and analyzed for p65, ß-actin and histone antibodies by western blot analysis. Blots in the figure were representative of three independent experiments. (K) The transfected NF-?B/Luciferase 293T cells were incubated with increasing concentrations of NNU219 or bortezomib for 6 h. Cells were then stimulated with 10 ng/ml of TNF-a for another 18 h. The activity of expressed luciferase was determined by using the Dual-Luciferase Reporter Assay System. (L) RPMI 8226 or ARH77 cell lines were treated with DMSO, IC50 of NNU219 or bortezomib for 12 h and harvested. Total RNA was isolated and subjected to qRT-PCR. The gene expression level of ICAM1, TNF-a, IL6 and VEGF was normalized to GAPDH using the 2-??CT method. In above experiments, the control group was incubated with the same concentration of DMSO in normal culture medium. Values were expressed as mean ± SD of triplicate samples of three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001). IL, interleukin; TNF, tumor necrosis factor; ICAM, intercellular adhesion molecule; VEGF, vascular endothelial growth factor; p-I?Ba, phosphorylated inhibitor of NF-?B; Con, control; MM, multiple myeloma.

Fig 3: NF-?B and mTOR activities orchestrate radiation-induced CXCL8 release.(A) Dot plots indicating means ± SD CXCL8 RNA levels relative to GAPDH in SK-Mel-28 cells transfected with p65 siRNA or nontarget siRNA 2 days after irradiation with 40 Gy. Indicated are two independent experiments with n = 4 separate transfections indicated as individual symbols (one symbol type for each experiment). (B to D) Dot plots indicating means ± SD CXCL8 concentrations in conditioned growth media of p65 knockout PANC-01 spheroids 7 days after irradiation (B) or SK-Mel-28 cells treated with the NF-?B inhibitor TPCA-1 (C) or rapamycin (D) 3 days after irradiation. Indicated are (B) n = 8 spheroids per condition of one representative of two independent experiments with different clones (second experiment in fig. S13C) or (C and D) n = 3 independent experiments as individual symbols. P values were calculated using unpaired (B) or paired t tests (A, C, and D) and repeated measures two-way ANOVA (D). (E and F) Representative images or dot plots of phospho-p70 expression in whole SK-Mel-28 cell lysates (E) or of I?B-a expression in subcellular fractions (F) was determined by WB. Top: Indicated is a representative WB. Bottom: Box plots indicating integrated density (ID) in WB images. For nuclear fractions, p65 IDs were divided by PCNA IDs. For cytosol fractions and whole-cell lysates, p65 IDs were divided by a-tubulin IDs. Indicated are n = 5 separate WBs from three independent experiments with each dot representing one WB. P values were calculated using paired t tests or a two-way ANOVA. KO, knockout; DMSO, dimethyl sulfoxide.

Fig 4: Cross-talk between mTOR signaling cascades, NF-?B, and CREB regulates the production of CCL5. HMC3 cells were treated with rapamycin (Rapa, 0-600nM) for 48 h, and the activation of NF-?B and mTOR signaling was assessed. (A) The total and phosphorylated levels of p65 subunit of NF-?B were analyzed by Western blotting, and GAPDH served as the loading control. (B) The total and phosphorylated levels of CREB were analyzed by Western blotting, and (C) active CREB (pCREB) protein level is further quantified (n=5 each group). (D) Transcriptional cross-talk between CREB, NF-?B, and mTOR pathway. NSC 228155 rescued the mTOR inhibitor (rapamycin)-mediated reduction of the gene expression level of CCL5, while p65-siRNA further counteracted NSC 228155’s stimulating effect on CCL5 expression. The gene expression level of the CCL5 was determined by qRT-PCR (n=3 each group). (E) NSC 228155 reversed the repressive effect of rapamycin on CCL5 production. The concentration of CCL5 protein in the cell culture medium was measured by ELISA (n=3 each group). (F) The total and phosphorylated levels of GSK-3ß were analyzed by Western blotting. Data are presented as mean ± SD. Statistical significance was determined by Student-t test and denoted by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 5: CDC20 induces proteasome-dependent degradation of p65 and CDC20 interacts with p65, related to Figs 3 and 4 A–DThe expression of CDC20 (A) and NF-?B pathway downstream genes IL-8, IL-6, and ICAM1 (B-D) of NC and CDC20si HEK293T cells after TNF-a stimulation determined by qRT–PCR (n = 6).EWestern blot analyses of the degradation of endogenous p65 protein in NC and CDC20si HEK293T cells.FWestern blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. HEK293T cells were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 µM MG132 (the proteasome inhibitor) for 6 h before collected.G, HCo-immunoprecipitation of endogenous CDC20 with endogenous p65 in hBMSCs.I, JCo-immunoprecipitation of endogenous CDC20 with endogenous p65 in mBMSCs.KThe co-localization of CDC20 and p65 in hBMSCs. Scale bar: 20 µm. Data information: Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey’s post hoc test and defined as ***P < 0.001.