Fig 2: Survival analysis with SurvExpress (n=468)A, B. Low expression of PTEN and PIK3C2A were correlated with high risk, poor prognosis and shorter OS time. C, D. High expression of ITPA and BCL3 indicated high risk, poor prognosis and shorter OS time. Kaplan-Meier survival curves were also constructed to reveal the relationship between predicted risk of ccRCC patients and the OS time. The results showed that patients with high risk had a significantly shorter OS time than those with low risk (A-D). Green and red lines indicated low- and high-risk groups, respectively. P <0.05 was considered to be statistically significant. Cens: Censored; Event: Death; Prog. Idx.: Prognosis Index.

Fig 3: Bcl3 binds to p52 with higher affinity than p50. Biolayer interferometry (BLI) binding analysis of recombinant His-tagged-Bcl3 to purified (A) p52:p52 and (B) p50:p50 homodimers. Each experiment was performed in duplicate, and one representative set of curves is shown. (C) Table showing the kinetic analysis in (A) and (B).

Fig 4: Bcl3 enhances the generation of the p52:p52 homodimer: p52:p52 vs. p52:RelA/p52:cRel/p52:p50. Diagrams illustrating the principle of multicolor BiFC assays: the p52:p52 homodimer formation brings VN and CN together to form a fluorescent protein which could be detected at the Venus channel; the (A) p52:RelA, (D) p52:cRel, and (G) p52:p50 heterodimer formation brings CC and CN together to form an intact Cerulean protein which could be detected using the Cerulean channel. The addition of RelB will heterodimerize with p52 or p50; therefore, both p52:p52 (Venus signal) and p52:p50 (Cerulean signal) will be decreased in (G). Fluorescent images of (B) p52:p52 vs. p52:RelA, (E) p52:p52 vs. p52:cRel, and (H) p52:p52 vs. p52:p50. The addition of Bcl3 enhanced p52:p52 homodimerization in all cases, as shown by the enhanced Venus signals; the addition of RelB decreased both p52:p52 (Venus) and p52:p50 (Cerulean) heterodimers in (H). (C,F,I) Quantification of the fluorescence intensity in (B), (E), and (H). The fluorescence intensity of p52-VN + p52-CC was determined as 100% for Venus, and the fluorescence intensity of p52-CC + RelB-CN, p52-CC + cRel-CN, or p52-CC + p50-CN was determined as 100% for Cerulean. The data were analyzed from images of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 versus no Bcl3 or RelB co-expression (t test). Error bars represent SD.

Fig 5: Bcl3 enhances the generation of the p52:p52 homodimer: p52:p52 vs. p52:RelB. (A) Diagram illustrating the principle of the multicolor BiFC assay. The p52:p52 homodimer formation brings VN and CN together to form a fluorescent protein which could be detected at the Venus channel; the p52:RelB heterodimer formation brings CC and CN together to form an intact Cerulean protein which could be detected using the Cerulean channel. (B) Fluorescent images with the co-expression of indicated plasmids. The addition of Bcl3 did not further enhance the fluorescence intensity of p52-VN and p52-CC. (C) Quantification of the fluorescence intensity in (B); the data were analyzed from images of three independent experiments. n.s., not significant versus no Bcl3 co-expression (t test). Error bars represent standard deviation (SD). (D) Fluorescent images with the co-expression of indicated plasmids. The addition of Bcl3 enhances p52:p52 homodimerization. (E) Quantification of the fluorescence intensity in (D), the fluorescence intensity of p52-VN + p52-CC was determined as 100% for Venus, and the fluorescence intensity of p52-CC + RelB-CN was determined as 100% for Cerulean. The data were analyzed from images of three independent experiments. *p < 0.05 versus no Bcl3 co-expression (t test). Error bars represent SD. (F) Co-IP showing less amount of p52 heterodimerized with RelB when there is an increasing amount of Bcl3 co-expressed.