Fig 1: Effects of FGF19 knockdown by small interfering RNA (siRNA) on protein phosphorylation. (a) Total cell lysates were collected 72 hours after indicated treatments, and analyzed for phosphorylation of FGF19, FRS2, FGFR4, AKT and ERK proteins. Anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody was included as a loading control. (b) Quantitative analysis of the changes in protein phosphorylation. Densitometry for Western blot signal was conducted, and intensity of the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. *P < 0.05. NC, sample treated with scramble siRNA as control; siRNA1,2,3, sample treated with siRNA1,2,3.
Fig 2: Effect of FGF19 knockdown by short hairpin RNA (shRNA) on cell growth in EPLC‐272H cells. (a) Relative FGF19 messenger RNA (mRNA) expression in cells was assessed 72 hours after FGF19 shRNA knockdown. EPLC‐272H cells engineered with inducible FGF19 shRNA and vector control (EGFP) were treated with doxycycline for 72 hours or left untreated. (b) Effect of FGF19 knockdown on cell proliferation. Cell proliferation was assessed with MTS proliferation assay. (c) Secreted FGF19 protein level in supernatant was analyzed by enzyme‐linked immunosorbent assay 72 hours after treatment with doxycycline or left untreated. Data represent the mean ± standard deviation; *P < 0.05. (d) Effects of FGF19 knockdown on FGF19 protein levels. Cells were treated with doxycycline or left untreated. Cell lysates were collected 72 hours post treatment and analyzed by Western blot.
Fig 3: Inhibitory effect of FGF19 knockdown on the growth of EPLC‐272H xenografts in vivo. (a) EPLC‐272H xenografts developed from EPLC‐272H cells, or cells carrying inducible engineered expression of tet‐controlled FGF19 short hairpin RNA (shRNA) (EPLC‐272H‐sh‐FGF19), or EGFP control (EPLC‐272H‐sh‐FGF19‐control) were randomized into indicated groups and treated with/without doxycycline for 14 days. (b) Immunohistochemical analysis was performed using anti Ki67, anti CC3, anti‐FGF19, and pAKT in tumor samples excised from xenografts of EPLC‐272H‐sh‐FGF19 treated with or without doxycycline. Ki67, CC3, FGF19, and pAKT analysis was conducted after 14 days of treatment. Quantification of positive signals was conducted using the Ariol system. Data represent the mean ± standard deviation.
Fig 4: FGF 19 messenger RNA (mRNA) upregulation and gene amplification in lung squamous cell carcinoma LSCC tumors. (a) FGF19 mRNA expression levels were analyzed in paired tumor and adjacent non‐tumorous tissues. Values were presented as log2 transformed relative fold changes in mRNA expression level compared to the paired non‐tumorous tissue. A twofold change threshold was set to identify obvious changes to gene expression. T, tumor tissue; N, non‐tumorous tissue. * represents tumor samples with gene amplification. (b) Representative images of FGF19 protein expression in LSCC tumor samples LSCC020, SCC023, LSCC038, and LSCC040. (c) Representative images of FGF 19 gene amplification in LSCC tumor samples LSCC001, LSCC023, LSCC028, and LSCC040. Fluorescence in situ hybridization (FISH) analysis was performed in tissue sections using probes against FGF 19 (red) and CEP 11 (green). Scale bars represent 10 μm for FISH images. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.
Fig 5: Characterization of lung squamous cell carcinoma (LSCC) tumor cells. (a) Representative images for fluorescence in situ hybridization (FISH) analysis staining with probes of FGF 19 (red) and CEP 11 (green) in EPLC‐272H and NCI‐H1703 cells. (b) FGF19 mRNA expression in EPLC‐272H, NCI‐H1703, and LSCC001 tumor tissues. Messenger RNA (mRNA) level of FGF19 was determined by quantitative real‐time‐PCR. The mRNA level of EPLC‐272H, and NCI‐H1703 was normalized to that of LSCC001 tumor tissue.
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