Fig 2: Apigenin inhibits cancer cell migration and inactivates the integrin ß1-FAK signaling pathway. Wound-healing assays were performed with or without 50–100 µM apigenin in HeLa (A) and C33A (B) cells for 0, 24, and 48 h. Left: representative images of scratches and recovery of wounded areas on cell monolayers at 0, 24, and 48 h after wounding. Right: semi-quantitative analysis of relative cell migration was performed according to the cells moving towards the scratched area at a certain time. Cell migration-related proteins p-FAK, paxillin, and integrin ß1 were detected in HeLa (C) and C33A (D) cells with or without 50 µM apigenin treatment for 24 and 48 h via Western blotting and quantified. Data are presented as the mean ± SD of at least three independent experiments. * and † p < 0.05 indicate significant differences as compared with the corresponding control or Api 50-treated groups. CON, 0.1% DMSO; Api 50, 50 µM apigenin; Api 100, 100 µM apigenin; Api50 24 h, 50 µM apigenin at 24 h; Api50 48 h, 50 µM apigenin at 48 h.

Fig 3: Schematic diagram of luteolin combined with asiatic acid’s anticancer molecular mechanism in cervical cancer. Lut combined with AsA mainly modulates FAK signaling (integrin ß1, paxillin and FAK) and PI3K/AKT signaling (PI3K, AKT and p70S6K) and causes JNK/p38 downregulation and ERK upregulation, inactivating or activating various signaling targets, such as Bcl2, Bax, mitROS, caspase-3 and RARP1; this leads to the induction of caspase-mediated intrinsic apoptosis, sub-G1 phase arrest and inhibition of cancer cell migration, which increase the anticancer effect on cervical cancer.

Fig 4: Cell adhesion and migration in response to the N-glycosylation of human and porcine fibronectin(A) Cells were pre-incubated with serum-free medium containing H2O (control), serum-free medium containing 0.5 mM EDTA, or culture medium containing 2 mM MnCl2 before plating on the indicated concentrations of fibronectin [FN] treated with (+) or without (-) PNGase F. Cells were plated on [FN] for 10 min and then their attachment was measured. Percentage of cells remaining attached on [FN] was shown. Data are the mean ± s.e.m. (n = number of independent experiments). *p < 0.05; **p<0.01; ***p < 0.001; NS, no significance. (B) Cell lysates from U2OS cells plated on 10 µg/ml FN treated with (+) or without (-) PNGase F for 10 min or 16h (overnight) were analyzed by Western blotting using antibodies against FAK, FAKpY397, total integrin ß1 (CD29), activated integrin ß1 (B44) and a-tubulin. Bottom: ratio indicates the expression of FAKpY397 normalized against FAK and activated integrin ß1 (B44) against to total integrin ß1 (CD29) from Western blotting. The ratio in the indicated conditions is calculated relative to that in the first lane in the same gel. Data are the mean ± s.e.m. (n = 5 independent experiments). NS, no significance. (C) Homo and porcine plasma fibronectin with (+) or without (-) a2-3,6,8 Neuraminidase (Sialidase) digestion were analyzed to assess the spectral counts of N-glycosylated residues with or without sialic acids at the various individually detected N-glycosylation sites. The plot shows the percentage of the spectral counts of the N-glycosylated residues with or without sialic acids relative to the total spectral counts for all detected N-glycosylation sites. The results demonstrated an efficient process for the trimming of sialic acids across the various N-glycosylation sites. (D) Cells in serum-free medium were plated for 20 min on [FN], treated with (+) or without (-) a2-3,6,8 Neuraminidase (Sialidase). The fold of cells remaining attached on [FN] relative to that on 0 µg/ml fibronectin is shown. Data are the mean ± s.e.m. (n = number of independent experiments). NS, no significance. (E) Cells in serum-free medium were seeded on plates coated with [FN], accompanied by ddH2O, AAL or WGA for 20 min. The fold of cells remaining attached on [FN] relative to that on 0 µg/ml fibronectin was measured. Data are the mean ± s.e.m. (n = number of independent experiments). *p < 0.05; NS, no significance. (F) Cells were plated on homo or porcine fibronectin, treated with (+) or without (-) PNGase F and then assayed for wound-healing migration. The percentage of wound closure at 10-h time point of wound-healing migration was calculated using Metamorph software and plotted. Data are the mean ± s.e.m. [homo: n = 5 (-PNGase F), n = 6 (+PNGase F) independent experiments; porcine: n = 6 (-PNGase F), n = 5 (+PNGase F) independent experiments]. **p<0.01; ***p < 0.001.

Fig 5: VEGFR2 protein expression is modulated through gene regulation by FAK in TNBC cells. (A) Knockdown of FAK decreased protein expressions of VEGFR2, p-FAK, and VEGF but knockdown of VEGFR2 did not affect these protein expressions in MDA-MB-231 and MDA-468 cells. (B) Protein expressions of VEGFR2, p-FAK, and VEGF were reduced in two stable clones of knockdown FAK MDA-Mb-231 cells. (C) VEGFR2 protein expressions in MDA-MB-231 and MDA-MB-468 cells were significant suppressed through PF-562271 treatment. (D) Overexpression of FAK could also increase VEGFR2, p-FAK, and VEGF protein expression in MDA-MB-231 and MDA-MB-468 cells.