Fig 2: Cell matrix adhesion properties are retained in FCPG mutants. (A) Scratch/wound healing assay was used to study cell spreading. Control (Fgfr1+/cKO;Fgfr2+/cKO) and Fgfr1FCPG/cKO;Fgfr2cKO/cKO primary FNP cells showed active spreading over 12 h (T12) in response to FGF1, PDGF-A, or serum. Fgfr1cKO/cKO;Fgfr2cKO/cKO mutant cells showed limited spreading upon FGF stimulation, but normal spreading in response to PDGF or serum. (B) Focal adhesion formation was assayed by Paxillin immunostaining in Fgfr1 signaling mutant primary FNP cells. GFP+ Fgfr1+/cKO;Fgfr2+/cKO;ROSA26mT/mG control, Fgfr1FCPG/cKO;Fgfr2+/cKO;ROSA26mT/mG, and Fgfr1cKO/cKO;Fgfr2cKO/cKO;ROSA26mT/mG mutant cells were treated with FGF1, PDGFA or serum for 3 h before analyzing Paxillin localization to focal adhesions. In control cells and Fgfr1FCPG/cKO;Fgfr2+/cKO, we detected multiple Paxillin+ foci upon FGF1, PDGFA, or serum stimulation. Fgfr1cKO/cKO;Fgfr2cKO/cKO;ROSA26mT/mG mutant FNP cells failed to form focal adhesion in response to FGF, in contrast to PDGF or serum (quantified in Supplemental Fig. S8A). (C) Focal adhesion formation was assayed by Paxillin immunostaining in Fgfr2 signaling mutant primary FNP cells. Cell spreading properties were analyzed for Fgfr2+/+ (Fgfr1CRISPR-KO;Fgfr2+/+), Fgfr2FCPG/FCPG (Fgfr1CRISPR-KO;Fgfr2FCPG/FCPG), Fgfr2-/- (Fgfr1CR-KO;Fgfr2CR-KO), and Fgfr2KD/KD (Fgfr1CR-KO;Fgfr2KD/KD) iFNP cells in which Fgfr1 was disrupted. Addition of FGF1, PDGFA, or serum resulted in robust cell spreading and formation of Paxillin+ focal adhesions in Fgfr2FCPG/FCPG and Fgfr2+/+ iFNP cells. Both Fgfr2-/- and Fgfr2KD/KD iFNP cells showed severe defects in focal adhesion formation upon FGF1 treatment, but were unaffected in response to PDGFA or serum (quantified in Supplemental Fig. S8D).

Fig 3: FGF2 upregulates ONECUT2 expression through the FGFR1/ERK/ELK1 signaling pathway.A The levels of ONECUT2 were detected by RT-PCR and western blotting after treatment with different concentrations of FGF2 (0, 5, 10, 20 ng/ml) for 24 h. B Luciferase reporter assay showing the promoter activity of ONECUT2 in PLC/PRF/5 cells and SNU398 cells after FGF2 treatment (20 ng/ml, 24 h). C The levels of ONECUT2 were detected in PLC/PRF/5 cells after FGF2 treatment (20 ng/ml, 24 h) and FGFR1, FGFR2, FGFR3, or FGFR4 knockdown by western blotting. D The relative luciferase activity was detected in PLC/PRF/5 cells after transfection with truncations and mutated ONECUT2 promoter constructs, followed by FGF2 treatment (20 ng/ml, 24 h). E HCC cells were transfected with ELK1-silencing lentivirus, followed by FGF2 treatment (20 ng/ml, 24 h). Luciferase reporter assay showing ONECUT2 promoter activity. F RT-PCR and western blotting showing ONECUT2 levels in HCC cells transfected with ELK1-silencing lentivirus followed by FGF2 treatment (20 ng/ml, 24 h). G Protein levels of ONECUT2, ERK, p-ERK, JNK, p-JNK, P38, p-P38, AKT, p-AKT, PKCa, and p-PKCa were measured by western blotting when PLC/PRF/5 cells were pretreated with inhibitors of ERK (SCH772984, 10 µM, 30 min), JNK (SP600125, 20 µM, 1 h), P38 (SB203580, 20 µM, 1 h), PI3K (LY294002, 20 µM, 1 h) or PKC (GO6983, 10 µM, 30 min), followed by administration of FGF2 (20 ng/ml, 24 h). H Relative binding of ELK1 to ONECUT2 promoter was determined by ChIP assays when HCC cells were treated with FGF2 (20 ng/ml, 24 h) and the indicated inhibitor. Data are mean ± SD. *P < 0.05, **P < 0.01.

Fig 4: GNA enhances the anti-tumor activity of erlotinib through FGFR in vitro.a H1650 cells, HCC827 cells, and HCC827ER cells were treated with various concentrations of GNA, erlotinib, or their combination for 24 h and were then probed with specified antibodies. b-e FGFR and the downstream proteins were analyzed by a western blot. HCC827ER cells were transfected with FGFR1 and FGFR2 siRNA for 48 h, then the medium was replaced and the cells were further incubated for 24 h. After incubation, the protein expression of FGFR1 (b) and FGFR2 (c) were analyzed by western blot, the mRNA expression of FGFR1 (d) and FGFR2 (e) were analyzed by real-time PCR. (f, g) FGFR1 or FGFR2 knockdown HCC827ER cells were treated by GNA for 72 h, and the cell viability was assessed using the CellTiter-Glo assay. The data are presented as the means ± SD and were analyzed by a one-way ANOVA, **P < 0.01, ***P < 0.001

Fig 5: FGF18 bound FGFR2 and FGFR3 to activate downstream ERK and Akt signaling. After knockdown of FGFR expression in Ishikawa cells (A), a few downstream target genes of FGF18-FGFR signaling were detected by WB assay (B). (C) FGFR2- and FGFR3-knockdown Ishikawa cells had reduced expression of phospho-ERK, phospho-Akt, Survivin and CD44V6; FGFR1- and FGFR4- knockdown cells had the same expression of phospho-ERK, phospho-AKT, Survivin and CD44V6 as the control group of si-NC Ishikawa cells; and the expression of ERK, Akt, and P53 was not different among all FGFR-knockdown groups. (D and E) After co-culturing FGFR2/4-knockdown Ishikawa cells and hESCs, the results showed that FGF18/si-FGFR2 signaling inhibited the proliferation and invasion of Ishikawa cells compared to FGF18/si-NC or FGF18/si-FGFR4 signaling (*P=0.05; **P=0.01; ***P=0.001).