Fig 2: NaB accelerated gp130 degradation by increasing the level of TRAF5. a TRAF5 transcription analyzed by the GEPIA web tool. b, c Western blotting of TRAF5 protein levels in cells treated with NaB (5 mM) for 24 h. d Co-immunoprecipitation assay of gp130 and TRAF5 in HT-29 cells treated with 5 mM NaB for 24 h. e–g Rescue experiments of HT-29 cells transfected with TRAF5 siRNA. h A schematic diagram of the mechanism

Fig 3: miR-141-3p targets TRAF5 and TRAF6. a Predictive target genes of miR-141-3p from TargetScan, miRanda and miRDB. b Western blotting of TRAF1, TRAF5 and TRAF6 expression in the indicated cells. α-Tubulin served as the loading control. c Predicted miR-141-3p targeting sequence and mutant sequences in 3’UTR s of TRAF5 and TRAF6. d-f Luciferase assay of cells transfected with pmirGLO-3’UTR reporter of TRAF5 and TRAF6 in the miR-141-3p overexpressing and silencing PCa cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. g-i MiRNP IP assay showing the association between miR-141-3p and TRAF5 and TRAF61transcripts in PCa cells cells. Pulldown of IgG antibody served as the negative control. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05

Fig 4: Clinical relevance of miR-141-3p with TRAF5, TRAF6 and NF-kB signaling activity in human PCa and bone tissues. a Analysis of miR-141-3p expression with TRAF5, TRAF6 and nuclear p65 in 4 non-bone metastatic PCa tissues (T1–4) and 4 bone metastatic PCa tissues (T5–8). U6 was used as the control for RNA loading. miR-141-3p expression levels were normalized to that miR-141-3p expression of sample one. Each bar represents the mean ± SD of three independent experiments. Loading controls were α-tubulin and p84 for the cytoplasmic and nuclear fractions. b-d Correlation between miR-141-3p levels and TRAF5, TRAF6 and nuclear p65 expression in PCa and bone tissues.The expression levels of TRAF5, TRAF6 and nuclear p65 were quantified by densitometry using Image J Software, and normalized to the levels of α-tubulin and p84, respectively. The sample 1 was used as a standard. The relative expressions of miR-141-3p and these proteins were used to perform the correlation analysis

Fig 5: TRAF expression under different experimental conditions. CA and AA HUVECs were incubated with TNF (30 ng/mL, 6 h), exposed to HSS (20 dyne/cm2, 24 h), or HSS (20 dyne/cm2, 24 h) followed by TNF (30 ng/mL, 6 h). HSS significantly upregulated TRAF3 expression (b) and downregulated TRAF5 (c). However, TRAF2 expression did not change across all conditions (a). Data are represented as mean ± SE from three independent experiments in three different cell lines per racial group. * p = 0.02 compared to other conditions; # p = 0.003 compared to control and TNF.