Fig 2: MMPs are required for glioma invasion and TWEAK-induced MMP9 expression is p52-dependent. (A) Quantification of invasion density at 48 h for BT116 cells with or without 10 ng/ml TWEAK in the collagen matrix. Cells were treated during the 48 h invasion assay with broad-spectrum MMP inhibitor GM6001 at indicated concentrations. Data represent average numbers of invading cells per 1-mm2 field (n = 3 wells) ± S.E.M. **p < 0.01 ***p < 0.001 between indicated groups using unpaired Student’s t-test. (B) qRT-PCR analysis of MMP mRNA expression in cultured BT116 cells. mRNA was collected at indicated time points after stimulation with TWEAK (10 ng/ml) or TNFa (10 ng/ml). Data represent average mRNA expression relative to GAPDH mRNA and normalized to untreated cells at t = 0 (n = 3 replicates) ± S.D. (C) Western blot of whole-cell lysates from cultured BT25, BT116, and U87 cells transduced with shRNA targeting p100/p52 (NFKB2) or a scrambled control (Control). Lysates were collected at 48 h after stimulation with 10 ng/ml TWEAK or vehicle control. White arrowhead indicates p52 band. Black arrowhead indicates non-specific band. (D) qRT-PCR analysis of MMP9 mRNA expression from cell lines in Figure 4C. mRNA was collected at 48 h after stimulation with 10 ng/ml TWEAK or vehicle control. Data represent average MMP9 mRNA expression relative to RPLP0 mRNA and normalized to untreated control (n = 3 replicates) ± S.D. ***p < 0.001 between indicated groups determined by unpaired Student’s t-test for each cell line.

Fig 3: miR-361-3p regulates CRC growth via the noncanonical NF-?B pathways. (A) Immunoblot analysis of TRAF3, NIK, p-IKKa/ß, p100, p52, p27, CDK4, Cyclin D1, Bcl-2, Bax, Caspase3, Cleaved caspase3 and ß-actin in CRC cells transfected with empty vector, miR-361-3p. (B) Immunoblot analysis of abovementioned proteins in CRC cells transfected with sh-NC, sh-TRAF3. (C) Immunoblot analysis of abovementioned proteins in CRC cells transfected miR-361-3p and co-transfected groups of miR-361-3p and TRAF3. (D) Proposed model of miR-361-3p-promoted proliferation via targeting TRAF3-activated the noncanonical NF-kB pathway in CRC cells

Fig 4: Concomitant CEACAM1 and p52 expression in human gastritis and tumor samples. Representative images of normal mucosa (n = 3), gastritis (n = 13), intestinal-type gastric cancer (GC) (n = 8), and diffuse-type GC (n = 3) tissue are shown, which were stained for CEACAM1 and p52 (NF-?B2). Scale bar: 100 µm.

Fig 5: Activation of the non-canonical NF-?B pathway upon H. pylori infection in MKN45 gastric cells. MKN45 cells were infected with P12 or G27 wild type H. pylori strains or isogenic mutant strains at an MOI of 10, 20 or 50 for 8 h. (a) p100 to p52 processing detected by Western blot. GAPDH was used as loading control. (b) IL8 secretion was measured by ELISA. (c) A20, (d) LTß and (e) CXCL10 and CCL2 mRNA levels were analyzed by qPCR. GAPDH was used as a housekeeping gene. All qPCR values were normalized to uninfected controls and calculated using the 2-??ct equation. Graphs show mean ± SD of at least three independent experiments. One-way ANOVA with Bonferroni correction (a,c–e) or Kruskal–Wallis with Dunn’s correction (b). * p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001.