Fig. 3: PUM1-TRAF3 expression activates the non-canonical NF-κB pathway and induces NF-κB2 and RelB translocation.

a Both 1196C and 1196PT cells with silenced TRAF3 gene expression (TRAF3 KO) showed increased proliferation (63.55 ± 3.65% in 1196C TRAF3 KO, 37.36 ± 1.22% to 68.72 ± 8.55% in 1196PT and 1196PT TRAF3 KO, respectively (***p < 0.001). Data presented as mean ± S.D. b Proteins differentially expressed in PUM1-TRAF3-transduced cells were analyzed; these included PUM1, TRAF3, PUM1-TRAF3, and NIK (***p < 0.001). The protein expression was assessed by area (***p < 0.001). Data presented as mean ± S.D. c PUM1-TRAF3-induced NF-κB activation with or without naïve TRAF3 results in the translocation of NF-κB2(p52) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (***p < 0.001). Data presented as mean ± S.D. d Immunofluorescence staining for NF-κB2 and RelB in SNU1196 control vector-transduced and SNU1196 PUM1-TRAF3-transduced cells. TRAF3 knockout enhances NF-κB2 and RelB translocation in control vector and PUM1-TRAF3-transduced cells. (DAPI, blue; Green, PT or C; Red, NF-κB2 or RelB). e PUM1-TRAF3-transduced and control vector (C)-transduced cells (5 × 10⁶ cells/site) with or without TRAF3 expression were injected subcutaneously into four groups of mice, and tumor size was measured every 7 days (volume = length × width² × 1/2). At 27 days post injection, the average volumes of tumors were 582.6 ± 223.6 mm³, 802.8 ± 167.0 mm³, 898.9 ± 349.7 mm³ (p < 0.05), and 1019.9 ± 156.6 mm³ (p < 0.01) in 1196C, 1196PT, 1196C TRAF3 KO, and 1196PT TRAF3 KO mice, respectively (*P < 0.05; **P < 0.01; ns no significance). Data presented as mean ± S.D. f Immunohistochemical staining of GFP (control vector or PUM1-TRAF3), NIK, NF-κB2(p52) and RelB in mouse tumor tissues in C- and PT-expressing cell lines.