Fig 2: CnAs negatively regulate LtßR- and TWEAK-dependent processing of p100 to p52.A. Effect of CnAa/ß depletion on LtßR-dependent processing of p100 to p52. Endogenous CnAa/ß were depleted in MEF cells by siRNA-mediated knockdown. CnAa-, CnAß-, or CnAa/ß-depleted MEFs were treated with the agonistic anti-LtßR antibody for 24 h (LtßR-Ab) or untreated (control). Total cell lysates were analyzed by western blotting. siRNA used for knockdown are indicated at the top of panels. Antibodies used for western blotting are indicated at the left of panels. Band intensities of p52 relative to Tubulin were normalized to that of control (control siRNA-treated cells without stimulation) and exhibited under panels. Blots are cropped for clarity. Full-length blots are presented in Supplementary Figure 2. B. Western blotting of total lysates of MEFs treated with TWEAK. Endogenous CnAa/ß were depleted in MEFs by siRNA-mediated knockdown. CnAa-, CnAß-, or CnAa/ß-depleted MEFs were treated with recombinant TWEAK for 3 h or untreated (control). Total cell lysates were analyzed by western blotting. siRNAs used for knockdown are indicated at the top of panels. Band intensities of p52 relative to Tubulin were normalized to that of control (control siRNA-treated cells without stimulation) and exhibited under panels. Antibodies used for western blotting are indicated at the left of panels. Blots are cropped for clarity. Full-length blots are presented in Supplementary Figure 2.

Fig 3: Plumbagin inhibits NIK activity and NF-?B2 p100 processing.A, bone marrow cells from C57BL6 mice were cultured with M-CSF for 3 days to expand macrophages. The cells were starved for 4 h followed by treatment with RANKL (R) plus vehicle or plumbagin (1 µM) for 15 and 60 min, respectively. Phosphorylated (p)-I?Ba was tested by Western blot (WB). B, NIK activity was tested in the presence of indicated dose of plumbagin using ADP-Glo Kinase Assay kit, four repeats each dose (one sample each dot). **p < 0.01 versus vehicle (0). C, macrophages, generated as in (A), were treated with RANKL in the presence of vehicle or plumbagin for 8 h. Cell lysates were used to test protein levels of p100/p52, RelB, RelA, and ß-actin by WB. D, CH310T1/2 mouse MSCs were treated with vehicle (V) or plumbagin for 8 h. P100/p52, RelB, and ß-actin proteins in the cell lysate were tested by WB. Each WB was repeated three times with similar results. NIK, NF-?B-inducing kinase; RANKL, receptor activator of nuclear factor kappa-B ligand.

Fig 4: Gene expression analysis of wild-type and p100-/- spleens.(A) Changes in mRNA levels of selected genes were analyzed by qRT-PCR using RNA samples isolated from spleens of four wild-type and four p100-/- animals. Data are expressed as mean values ± SD. Differences were analyzed by Welch tests. P=0.05 was considered significant. All genes shown were significantly differentially regulated in wild-type compared to mutant mice. (B) Western blot analysis of total protein extracts from wild-type and p100-/- spleens (30 µg/sample; protein extracts from three independent spleens are shown for each genotype) for the presence of p100/p52, ENPP2/ATX, and TRAF1. p52 wild-type protein and p52 protein resulting from the knock-in of the stop codon migrated with different speed due to a few amino acids difference in length. The asterisk indicates an unspecific signal. The membrane was probed for ß-actin as a loading control.

Fig 5: Inhibition of miR-302d reversed p100 attenuation of Cyclin D1 protein expression, anchorage-independent growth, and promoted G0/G1 cell-cycle progression(A–D) UMUC3(p100) cells and p100-/- (p100) cells were stably transfected with construct of anti-miR-302d and the miR-302d were determined by quantitative real-time PCR. The symbol (*) indicates a significant inhibition of miR-302d expression as compared with vector transfectant (p < 0.05) (A and B). The cyclin d1 3'UTR luciferase reporter was transiently transfected into indicated cells and luciferase activity of each transfectant was evaluated. The results were presented as relative cyclin D1 3'-UTR activity and the symbol (*) indicates a significant increase in cyclin D1 3'-UTR luciferase activity as compared with that in vector transfectant (p < 0.05) (C and D). (E and F) The cell extracts were subjected to Western Blot and ß-Actin was used as a protein loading control. (G) Flow-cytometry analysis of cell cycle was performed as indicated. (H and I) Anchorage-independent growth was determined in soft agar. And the number of colonies was scored and presented as colonies per ten thousand cells. The symbol (*) indicates a significant increases in comparison to the vector transfectant (p < 0.05).