Fig 1: Disruption of the TRIB3‒GSK-3β interaction suppresses profibrotic AMs by restoring A20 activity. (A) I-TASSER server for analyzing the secondary structures of TRIB3-binding regions in GSK-3β and the amino acid sequences of the helical peptides in GSK-3β. (B) Surface plasmon resonance for analyzing the kinetic interaction between TRIB3 and the α-helical peptide GA. (C) IP assay for determining the interaction between TRIB3 and GSK-3β in the presence of PGA. (D) IP assay for evaluating A20 phosphorylation in the presence of PGA. (E) A20 activity was measured in AMs treated with PGA. (F) Immunoblot analysis of C/EBPβ protein levels in AMs after PGA treatment. The loading control was GAPDH (n = 3). (G) Immunoblot analysis of GSK-3β protein levels in AMs after PGA treatment. The loading control was GAPDH (n = 3). (H) Quantitative mRNA analysis of Gsk3b expression in AMs from PF mice after PGA treatment (n = 3). (I) Quantitative mRNA analysis of Trib3 expression in AMs from PF mice after PGA treatment (n = 3). (J) Immunostaining revealing the expression of CD206 in PGA-treated AMs. (K) Quantitative mRNA analysis of the target genes in AMs from PF mice after different treatments (n = 3). (L) The experimental scheme for determining the cross-talk between AMs from PF mice and primary lung fibroblasts. (M, N) The antifibrotic effect of PGA was verified based on α-SMA expression in fibroblasts (M, n = 3) and fibroblast contraction in 3D collagen matrices (N, n = 4). (B–K, M, and N) Representatives of 2–4 experiments. Data are represented as mean ± SEM (n = 3–4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar: 8 μm (J), 2 mm (M), and 50 μm (N).
Fig 2: Tribbles pseudokinase 3 (TRIB3) promoted β‐catenin/Wnt signaling. A, Western blotting of β‐catenin and Wnt3A in HCT116/CT26 cells treated with PBS, transforming growth factor‐β (TGF‐β, 100 ng/mL), TGF‐β (100 ng/mL) combined with GN44028 (20 nmol/L) (T+G), or TGF‐β (100 ng/mL) combined with TRIB3 siRNA (T+Si#1 and T+S#2). B, Colony formation rates of TGF‐β (100 ng/mL) cultured CT26/HCT116 cells treated with PBS or PNU‐74654 (50 μmol/L). C, Tumorigenesis capability of TGF‐β (100 ng/mL) cultured CT26/HCT116 cells treated with PBS or PNU‐74654 (50 μmol/L). D, Relative migrating cell numbers of TGF‐β (100 ng/mL) cultured CT26/HCT116 treated with PBS or PNU‐74654 (50 μmol/L). E, Immunofluorescence staining of Wnt3A in tumor tissues from metastasis (M) and nonmetastasis (N) colorectal cancer patients. Intensity of β‐catenin/Wnt expression was determined in 30 patients. Scale bar, 50 μm. *P < .05. **P < .01
Fig 3: TRIB3 activates the AKT signaling pathway. TRIB3 overexpression activated the AKT signaling pathway in (A) HSC3 and (B) Cal27 cells. TRIB3 knockdown inhibited the AKT signaling pathway in (C) SCC9 and (D) SCC15 cells. TRIB3, tribbles pseudokinase 3; p, phosphorylated; SCR, scrambled; shRNA, short hairpin RNA.
Fig 4: RCC cell proliferation, migration and invasion assays. A: CCK-8 assays show that TRIB3 overexpression significantly promotes the proliferation of 786-O (P = 0.0188) and Caki-1 cells (P = 0.009). B: A decreased proliferation rate was detected in TRIB3 knockdown cells compared with that in nontargeting control cells (P < 0.05). C/E: Wound healing assays and Matrigel-free transwell assays indicate that overexpression of TRIB3 promotes the migration of 786-O and Caki-1 cells (P < 0.05). D/F: Downregulation of TRIB3 also inhibits the migration of 769-P and A498 cells (P < 0.01). G: In Matrigel-coated transwell assays, overexpression of TRIB3 markedly increases the invasive ability of 786-O and Caki-1 cells (P < 0.01). H: TRIB3 knockdown decreases the invasive potential of 769-P and A498 cells (P < 0.05). “ * ” represents P < 0.05, “ ** ” represents P < 0.01, “ *** ” represents P < 0.001.
Fig 5: Increased expression of TRIB3 in LIHC tissues and this was associated with poor prognosis. (A) The expression of TRIB3 in different tumors; (B) in TCGA dataset, the expression of TRIB3 in LIHC tumor tissue was higher than that in normal tissues; (C,D) two datasets from Oncomine showed that TRIB3 is highly expressed in tumor tissues of HCCs; (E,F) K-M plot analysis showed that TRIB3 is related to the OS of HCC patients (data obtained from GEPIA and K-M plotter databases). **, P<0.01. TRIB3, tribble pseudokinase 3; LIHC, liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; HCCs, hepatocellular carcinomas; K-M, Kaplan-Meier; OS, overall survival.
Supplier Page from Abcam for Anti-TRIB3 antibody