Fig 1: PCBP3 posttranscriptionally increases PFKFB3 expression by promoting its translation during myofibroblast activation. (A) PFKFB3 degradation in mouse lung fibroblasts when protein synthesis was inhibited by 50 µM cycloheximide (mean ± SD, n = 3). (B) Prediction of RNA–protein interaction of PFKFB3 mRNA with PCBP3 protein using the catRAPID algorithm. Red represents interaction strength. (C) Western blot analysis of PFKFB3 and PCBP3 protein levels in mouse lung fibroblasts stimulated with TGF-ß1 for the indicated concentrations. (D) Quantification of PFKFB3 and PCBP3 protein levels relative to ß-actin is shown (mean ± SD, n = 3, **p < 0.01, ***p < 0.001 compared with 0 by one-way ANOVA). (E) Quantitative RT-PCR (qPCR) to show the effect of PCBP3 overexpressing on PFKFB3 RNA immunoprecipitation (RIP) in mouse lung fibroblasts. Values were plotted as mean ± SD from three independent experiments. p value was calculated by Student t test. ***p < 0.001. (F) Mouse lung fibroblasts expressing Flag-PCBP3 were fractionated into cytoplasmic extracts through sucrose gradients. The arrow indicates the direction of sedimentation. The distribution of PFKFB3 and ß-actin mRNAs was quantified by RT-PCR analysis of RNA isolated from 12 gradient fractions. Statistical analyses were performed using Student t test. ***p < 0.001. (G) Mouse lung fibroblasts were transfected with Flag-PCBP3, and then transfected with PFKFB3-siRNAs or NC-siRNA. The levels of Fibronectin?Collagen I?PFKFB3?a-SMA?PCBP3 and ß-actin assessed by western blot. (H) Graphical representation of the relative levels of indicated proteins (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA).
Fig 2: Anlotinib represses PCBP3 expression levels during myofibroblast activation. (A) Immunofluorescence for PCBP3 (green) in mouse lung fibroblasts treated with anlotinib for 3 h, followed by TGF-ß1 for an additional 24 h. DAPI-stained nuclei (blue). Scale bar, 25 µm. (B) Western blots analysis of PCBP3 and ß-actin in primary mouse lung fibroblasts. (C) Quantification for the indicated proteins (mean ± SD, n = 3). (D) The Western blots analysis of PCBP3 and ß-actin in IMR90 cells treated with anlotinib for 3 h, followed by TGF-ß1 for an additional 24 h. (E) Quantification for the indicated proteins (mean ± SD, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 VS TGF-ß1-treated group by ANOVA.
Fig 3: Schematic representation of PCBP3-PFKFB3-dependent glycolysis and the inhibitory effect of anlotinib on this pathway. Lung injury induces PCBP3 expression, which results in an increase in PFKFB3 expression by promoting its translation, resulting in the augmentation of glycolysis in lung fibroblasts. Glycolytic reprogramming participates in myofibroblast activation and furthers lung fibrosis. The tyrosine kinase inhibitor anlotinib inhibits PFKFB3-driven glycolysis by decreasing the expression of PCBP3, thereby suppressing myofibroblast activation and inhibiting the exacerbation of lung fibrosis.
Fig 4: Anlotinib decreases PCBP3 expression and inhibit the PFKFB3-driven glycolysis in fibrotic rodent lungs. (A) Intervention dosing regimen of anlotinib in experimental mouse model of fibrosis. C57BL/6 mice were intraperitonealy injuected with 1 mg/kg of anlotinib or vehicle (n = 5-6 per group) daily after bleomycin instillation. Lungs were harvested at 21 days. Western blot analysis of PCBP3, ß-actin was used as a loading control (n = 6). (B) Quantification of PCBP3 protein levels relative to ß-actin is shown (mean ± SD, n = 6). (C) Representative images show PCBP3 staining of lung sections from the indicated groups of mice. Scale bars, 100 µm. (D) lactate contents in lung tissues from mice (mean ± SD, n = 5). (E) Western blot analysis of PFKFB3, ß-actin was used as a loading control (n = 6). (F) Quantification of PFKFB3 protein levels relative to ß-actin is shown (mean ± SD, n = 6). (G) Representative images show PFKFB3 staining of lung sections from the indicated groups of mice. Scale bars, 100 µm. **p < 0.01, ***p < 0.001 VS BLM-treated group by one-way ANOVA.
Supplier Page from Abcam for Anti-PCBP3 antibody