Fig 2: Expression level of total focal adhesion kinase (tFAK) and phosphorylated FAK (pFAK) with inhibition of FAK. (A and B) Time-dependent effect of the FAK inhibitor Y15 on the expression of tFAK and pFAK in endothelial cells and hepatoblastoma cells, respectively, within 2 h. Quantification of pFAK in tFAK expression by image analysis of the western blot bands in (C) endothelial cells and (D) hepatoblastoma cells. Values represent the mean ± standard deviation, n=4. *P<0.05 denotes a statistically significant difference compared with the other groups. (E) Double-labeled immunofluorescence analyzed the effects of distribution and expression of pFAK (green) and tFAK (red) proteins with inhibition of FAK after 5 min and 2 h (blue, DAPI stained nucleus); scale bar, 20 µm. (F) Enlarged images of designated regions, labeled by white square frames in (E); the green arrows indicate clear distribution of pFAK at the leading edge of the cells, while FAK with red fluorescence occupied the whole cytoplasm with 2 h FAK inhibition. A lower degree of green fluorescence may be observed at the cell periphery; scale bar, 2.5 µm.

Fig 3: C1GALT1 is involved in the radioresistance of esophageal cancer cells via regulating ß1-integrin signaling. (A) The p-FAK (Tyr397) expression was measured by western blot. (B) ECa109 cells were pre-incubated with 2 mg/ml ß1-integrin-blocking antibody (P4C10) for 1 h. The expression of p-FAK (Tyr397) and p-Akt (Ser473) was analyzed. (C) ECa109 cells were pre-incubated with 10 mmol/L FAK inhibitor for 1 h. The p-FAK and p-Akt expression levels were analyzed. (D) ECa109 cells were pre-incubated with P4C10 or FAK inhibitor for 1 h followed by 4 Gy X-ray irradiation. The apoptosis of cells was determined by flow cytometry 48 h after irradiation. (E) Schematic representation of the proposed molecular mechanism of C1GALT1.

Fig 4: Inhibition of focal adhesion kinase (FAK) induced phosphatidylinositol 3-kinase (PI3K), AKT and phosphorylated AKT (pAKT at Thr308) in the two cell lines within 2 h. Expression level of PI3K by western blot assays and quantification of PI3K expression level by image analysis of the western blot bands within 2 h in (A) endothelial cells and (B) hepatoblastoma cells, respectively, with inhibition of FAK. The expression level of ß-actin in each group was used as an internal control, and relative expression level were calculated. Expression level of pAkt by western blot assays and quantification of pAKT in total AKT (tAKT) expression by image analysis of the western blot bands in (C) endothelial cells and (D) hepatoblastoma cells. *P<0.05 denotes a statistically significant difference. (E) Immunofluorescence analysis of the effects of the distribution and expression of pAKT (red) proteins with inhibition of FAK at 5 min and 2 h (blue, DAPI-stained nuclei); scale bar, 20 µm. DAPI, 4',6-diamidino-2-phenylindole.

Fig 5: Fangchinoline suppresses the growth and metastasis potential of A375 cells by inhibiting the phosphorylation of FAK (A) A375 cells were treated with DMSO, PF-562271 or indicated concentrations of fangchinoline, cells growth was detected by MTT assay. Data are shown as mean ± SEM. (B) A375 cells were treated with DMSO, PF-562271 or indicated concentrations of fangchinoline for 24 h; Transwell assay without Matrigel was performed. Cells were counted and results represent the mean ± SD of three experiments. **P<0.01 vs. DMSO treated group. (C and D) A375 cells were treated with DMSO, PF-562271 or indicated concentrations of fangchinoline for 24 h; the indicated proteins and mRNA levels were detected by western blot analysis and real-time PCR. Data are shown as mean ± SEM. **P<0.01 vs. DMSO treated group.