anti primary fgfr2(23328s)  (Cell Signaling Technology Inc)

Journal: Pharmaceuticals

Article Title: Inhibition of FGFR2 Signaling by Cynaroside Attenuates Liver Fibrosis

doi: 10.3390/ph16040548

Figure Lengend Snippet: High expression of fibroblast growth factor receptor 2 (FGFR2) coincides with liver fibrosis. ( A ) The expression of FGFR2 in fibrotic liver and normal liver tissues was evaluated through data mining of human samples affected by Hepatitis B infection (GSE38941), alcohol abuse (GSE28619), and nonalcoholic steatohepatitis (GSE48452), as well as mouse samples induced with carbon tetrachloride (CCl 4 ) (GSE152329, GSE98577). The differential fold expression was determined by comparing the expression of FGFR2 in the fibrotic liver and normal liver tissues. ( B ) A comparison of changes in FGFR2 expression in liver fibrosis patients in remission (improved) or not in remission (not improved) with mining data of GSE175448. ( C ) The correlation between FGFR2 expression and the liver fibrosis score (Ishak score) was analyzed by comparing FGFR2 expression in three pairs of liver fibrotic tissues and normal liver tissues through immunohistochemistry. ( D ) FGFR2 expression in liver fibrosis tissues and normal liver tissues was verified by Western blot and ( E ) q-PCR analyses. ( E ) Liver tissues from mice treated with CCl 4 for different durations were used to determine the expression of the liver fibrosis markers Actin Alpha 2 (ACTA2) and FGFR2 by q-PCR and the trends of their expression with increasing days of induction. ( F ) The expression of ACTA2 and ( G ) FGFR2 was determined by q-PCR in liver tissues from mice treated with CCl 4 for varying durations. The statistical significance was determined based on the p -value. The results are marked as significant “*” when p < 0.05, “**” when p < 0.01, “***” when p < 0.001, and not significant (ns) if p ≥ 0.05.

Article Snippet: The Western blot and IHC analyses were performed using primary antibodies against FGFR2 (23328S, CST, Danvers, MA, USA), phospho-FGFRs (AF3285, R&D, Minneapolis, MN, USA), α-SMA (ab7817, Abcam, Waltham, MA, USA), FAP (AF3715, R&D, Minneapolis, MN, USA), and β-actin (MAB8929, R&D, Minneapolis, MN, USA).

Techniques: Expressing, Infection, Comparison, Immunohistochemistry, Western Blot, IF-P

Journal: Pharmaceuticals

Article Title: Inhibition of FGFR2 Signaling by Cynaroside Attenuates Liver Fibrosis

doi: 10.3390/ph16040548

Figure Lengend Snippet: FGFR2 drives the process of liver fibrosis. ( A ) The expression of liver fibrosis markers (ACTA2, fibroblast activation protein alpha (FAP), alpha-1 type I collagen (COL1A1)) was measured by qPCR. The samples were divided into four groups according to whether cells were induced with or without TGF-β and whether cells were overexpressed by FGFR2. ( B ) The expression of markers in wild-type and FGFR2-OE cell lines under TGF-β induction was analyzed by Western blot analysis. ( C ) The expression levels of α-SMA and ( D ) collagen secretion in LX-2 and Huh-7 were evaluated, when FGFR2 was overexpressed or knocked down upon equal TGF-β induction. ( E ) Cells were collected after 48 h of co-culture, and the expression of α-SMA and ( F ) type I collagen in the lower layer cells was determined by ELISA. The results are marked as significant “*” when p < 0.05, “**” when p < 0.01, and not significant (ns) if p ≥ 0.05.

Article Snippet: The Western blot and IHC analyses were performed using primary antibodies against FGFR2 (23328S, CST, Danvers, MA, USA), phospho-FGFRs (AF3285, R&D, Minneapolis, MN, USA), α-SMA (ab7817, Abcam, Waltham, MA, USA), FAP (AF3715, R&D, Minneapolis, MN, USA), and β-actin (MAB8929, R&D, Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Western Blot, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, IF-P

Journal: Pharmaceuticals

Article Title: Inhibition of FGFR2 Signaling by Cynaroside Attenuates Liver Fibrosis

doi: 10.3390/ph16040548

Figure Lengend Snippet: CYN blocks the activity of FGFR2. ( A ) A total of 2081 natural compounds were screened through high-throughput affinity analysis chips, resulting in the identification of compounds capable of binding to the FGFR2 kinase domain. The affinity and p -value of each compound were represented, and the candidate compound CYN was located in the high-affinity region. ( B ) The binding signal of FGFR2 and CYN was analyzed using surface plasmon resonance (SPR) technology at five different concentrations, and the affinity constant K D was calculated by fitting the curve algorithm. ( C ) An in vitro FGFR2 catalytic system was established, and the kinase activity was compared by measuring the phosphorylation product generation and ( D ) ATP consumption during the catalytic process. A series of concentration gradients were set up to evaluate the correlation between the inhibitory effect of CYN and its concentration. ( E ) Computational docking was utilized to predict the binding site of CYN on FGFR2. The surface charges on the model are depicted in blue (negative) to red (positive), and the appearance of the compound CYN is represented in green. Noncovalent amino acids that provide binding for the compound are marked in red on the right figure. ( F ) The diagram depicts the binding of the CYN and FGFR2 amino acid residues. ( G ) Molecular dynamics (MD)simulations were conducted to compare the stability of FGFR2 protein and FGFR2-CYN complex in aqueous environments. The mean value of the root-mean-square deviation (RMSD)at the plateau period is marked by a dotted line. ( H ) SPR was used to measure the affinity between the CYN and FGFR2 proteins with single-point mutations. The affinity is ranked from strong to weak in the legend, and the right-side values represent the corresponding K D values. A higher K D value represents weaker binding of the mutated FGFR2 protein with CYN, indicating that CYN’s binding is more dependent on the mutated amino acid. The results are marked as significant “*” when p < 0.05, “**” when p < 0.01, “***” when p < 0.001, and not significant (ns) if p ≥ 0.05.

Article Snippet: The Western blot and IHC analyses were performed using primary antibodies against FGFR2 (23328S, CST, Danvers, MA, USA), phospho-FGFRs (AF3285, R&D, Minneapolis, MN, USA), α-SMA (ab7817, Abcam, Waltham, MA, USA), FAP (AF3715, R&D, Minneapolis, MN, USA), and β-actin (MAB8929, R&D, Minneapolis, MN, USA).

Techniques: Activity Assay, High Throughput Screening Assay, Binding Assay, SPR Assay, In Vitro, Phospho-proteomics, Concentration Assay, IF-P

Journal: Pharmaceuticals

Article Title: Inhibition of FGFR2 Signaling by Cynaroside Attenuates Liver Fibrosis

doi: 10.3390/ph16040548

Figure Lengend Snippet: CYN blocks the activation and development of liver fibrosis in vitro. ( A ) Evaluation of the inhibitory effect of CYN on the fibrosis-promoting effect of FGFR2. The fibrotic transformation of wild-type and FGFR2-OE LX-2 cells and Huh-7 cells was induced through TGF-β activation, followed by an intervention with CYN for the relevant groups. The expression changes of the fibrosis markers ACTA2 and COL1A1 were analyzed by qPCR. ( B ) Wild-type cell lines were employed in the aforementioned experiments, and the activation of FGFR2 was triggered by supplementation with the exogenous basic fibroblast growth factor (bFGF) factor. ( C ) Analysis of the extent of antagonism of CYN towards TGF-β Signaling. Activation induction models were established by adding or not adding TGF-β to the cell culture environment with or without the CYN intervention, and the expression of liver fibrosis markers was determined by qPCR, ( D ) Western blot, and ( E , F ) ELISA analyses. ( G ) A co-culture model was used to evaluate the blocking effect of CYN on liver fibrosis activation of signaling transmission. The activation intensity of lower-layer wild-type cells was measured and compared using α-SMA expression and collagen secretion. The results are marked as significant “*” when p < 0.05, “**” when p < 0.01, and not significant (ns) if p ≥ 0.05.

Article Snippet: The Western blot and IHC analyses were performed using primary antibodies against FGFR2 (23328S, CST, Danvers, MA, USA), phospho-FGFRs (AF3285, R&D, Minneapolis, MN, USA), α-SMA (ab7817, Abcam, Waltham, MA, USA), FAP (AF3715, R&D, Minneapolis, MN, USA), and β-actin (MAB8929, R&D, Minneapolis, MN, USA).

Techniques: Activation Assay, In Vitro, Transformation Assay, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Blocking Assay, Transmission Assay, IF-P