Fig 2: AGE-induced FAK activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced FAK phosphorylation. ECs were pretreated with PP2 (15 μmol/L) for 90 min before exposed to 100 μg/mL AGEs for 1 h. (b) Pretreatment of Src siRNA prevented AGE-induced FAK phosphorylation. ECs were pretreated with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c,d) Effects of K298M and Y530F on AGE-induced FAK phosphorylation. ECs were pretreated with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. FAK and its phosphorylation form were detected by western blotting. (e) AGE induced Src/FAK association in HUVECs. HUVECs were treated with 100 μg/mL AGEs for 1 h and lysed, and co-immunoprecipitation (co-IP) for FAK was performed. Increased expression of Src was detected by western blotting (upper panel). HUVECs were treated with 100 μg/mL AGEs for 1 h and co-IP for Src was performed. Increased expression of FAK was detected by western blotting (lower panel). (f) PF573228 prevented AGE-induced hyper-permeability of endothelial cells. HUVECs were treated with PF573228 (20 μmol/L) for 60 min before exposed to 100 μg/mL AGEs for 8 h. Culture medium was used as control. Permeability coefficient of the transflux of trace FITC-dextran (Pd) and TER were measured. The ratio of immunointensity between the phosphorylation of FAK (p-FAK 925) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs group or Mock + AGEs.

Fig 3: UNC119 KD in HT-29 CRC cells inhibits SFK activity and cell proliferation. a Representative western blot showing UNC119 expression level, total SFK (tSFK), total FAK (tFAK), tubulin (as loading control) and SFK phosphorylated on Y419 (pY419-SFK) and FAK phosphorylated on Y925 (pY925-FAK) in HT-29 cells expressing Cas9 and a doxycycline-inducible sgRNA vector, containing either a non-targeting guide RNA (c), or UNC119A (a) or UNC119B (b) targeting guide RNAs, induced with doxycycline for 72 h. b Bar graph depicting the mean change in expression upon sgRNA induction for pYSrc/tSrc, pYFAK/tFAK and UNC119, compared to the non-targeting sgRNA control (n = 5 independent experiments; data are mean ± SE. P values determined by one-way ANOVA with Bonferroni post hoc test). c HT-29 cells were transfected with UNCA-mCh or UNCB-mCh and probed for SFK phosphorylation by western blotting. Plot depicts pYSFK/tSFK normalised to non-transfected control against UNC119-mCherry expression normalised to GAPDH loading control from western blots (see also Supplementary Fig. 8b). d Fluorescence micrographs of HT-29 Cas9 cells expressing the doxycycline-inducible UNC119B sgRNA and treated with or without doxycycline for 72 h, transfected with Src-mCh, Yes-mCh or Fyn-mKate2 (see also Supplementary Fig. 8c). Dot plot depicts the endomembrane/total integrated fluorescence intensity for each SFK (n = 28 or 29 cells for Src-mCh, n = 28 or 34 cells for Yes-mCh and n = 12 or 14 cells for Fyn-mKate2; data are mean ± SD from two independent experiments, ***P < 0.001; ****P < 0.0001, as determined by Student’s t-test). e Clonogenic assay of HT-29 Cas9 cells containing the indicated sgRNAs and induced with doxycycline for the duration of the assay. f Bar graph depicting the mean colony area coverage upon induction of sgRNA, normalised to the non-targeting sgRNA control well. g Bar graph depicting the mean colony number change upon induction of sgRNA, normalised to the pre-doxycycline control wells. h Bar graph depicting the mean individual colony size change upon induction of sgRNA, normalised to the pre-doxycycline control wells (n = 4 independent experiments; data are mean ± SE; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant as determined by one-way ANOVA with Bonferroni post hoc test). Scale bars, 10 μm

Fig 4: Overexpression of CASC2 inhibits pancreatic cancer cell growth and progression partially by altering cell-cell adhesion. Overexpression of CASC2 suppresses (A) proliferation of AsPC-1 and PANC-1 cells, as determined by MTT assay, (B) cell colony formation of AsPC-1 and PANC-1 cells, as determined by colony formation assay, (C) migration of AsPC-1 and PANC-1 cells, as determined by wound healing assay (×40 magnification), and (D) invasion of AsPC-1 and PANC-1 cells, as determined by Transwell assay (×100 magnification). (E) Overexpression of CASC2 promotes apoptosis of AsPC-1 and PANC-1 cells, as determined by flow cytometry. (F) Western blot analysis of indicated proteins in AsPC-1 and PANC-1 cells overexpressing CASC2. (G) Morphology of AsPC-1 and PANC-1 cells transfected with CASC2 overexpression vector; scale bar, 50 µm. *P<0.05 and **P<0.01. cad, cadherin; CASC2, cancer susceptibility candidate 2; FAK, focal adhesion kinase; ITGB4, Integrin β4; MMP, matrix metalloproteinase; NC, negative control; OD, optical density; p-, phosphorylated.

Fig 5: Reduced cell spreading and decreased phosphorylation of FAK in THP1 cells with decreased expression of EPHB2. (A) CCR2 and ITGB1 expression in control and EPHB2 knockdown monocytes. Results are relative to mock control cells, set as 1. Mean ± sem of n = 3. * P < 0.05. (B) Quantification of ITGB1 immunoblot expressed as fold change compared to control THP1 cells and corrected for GAPDH. (C and D) Representative pictures (C) and quantification (D) of the cell surface area of adhered control and EPHB2 knockdown THP1 cells. Results are relative to mock control cells, set as 1. Mean ± sem of n = 3. * P < 0.05. (E and F) Immunoblots (E) and quantification (F) of phospho‐ and total P38, P42‐44, AKT, FAK (Y397 and Y925), and GAPDH in control cells and EPHB2 knockdown cells. (G and H) Immunoblots (G) and quantification (H) of phospho‐ and total FAK (Y397 and Y925) and GAPDH in control cells and EphrinB knockdown cells. (F and H) Expression is expressed as fold change compared to mock THP1 cells and is corrected for total protein expression. Mean ± sem of n = 3. * P < 0.05. (I) Schematic diagram of the effect of EPHB2 and EphrinB expression on FAK phosphorylation in monocytes