Fig 1: Effect of inhibition of EGFR and FGFR on PD-L1 expression in cancer cells. (A) Impact of K-ras on expression of PD-L1, EGF, EGFR, FGF1 and FGFR1 mRNA in HPNE cells, measured by qRT-PCR analysis. (B) Expression of PD-L1, FGFR1, and EGFR in HPNE cells stably transfected with K-ras (HPNE/K) in comparison with their parental HPNE cells. Protein levels were measured by immunoblotting analysis. Data are representative of three separate experiments. (C–D) Four human pancreatic cell lines were incubated with FGF1 and FGF2 (50 ng/ml). Cell surface PD-L1 and PD-L2 expressions were quantified by FACS analysis. (E) Four human pancreatic cell lines were incubated with FGF1 and FGF2 (50 ng/ml). Expressions of PD-L1 and PD-L2 were measured by immunoblotting. (F) Expressions of PD-L1 and FGFR1, in Panc-1 and SW1990 FGFR1 KO cells, were measured by immunoblotting. (G-H) PD-L1 cell surface expression in Panc-1 and SW1990 FGFR1 KO cells was quantified by FACS analysis. (I-J) Correlation between PD-L1 and FGFR1 expression in human pancreatic tumor samples (n=81) using tissue microarray. The protein expression was determined by immunohistochemistry staining. Representative images of immunostaining and H/E staining (scale bars, 50 μm) are shown in (I); the quantitative data are shown in (J). Data are means ± SEM of three separate experiments; Two-tailed unpaired t-test for A, C-D; One-way ANOVA followed by Tukey post hoc test for G-H. Spearman’s rank correlation test for J. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 2: Regulation of PD-L1 expression by growth factor signaling. (A) HEK293T were incubated for 24 h with doxycycline (Doxy), or with conditioned medium (CM) from T-Rex/K-ras cells incubated with (ON) or without (OFF) doxycycline. PD-L1 mRNA was measured by qRT-PCR. (B) EGF, EGFR, FGF1, FGFR1, IL-1α and IL1R1A mRNA levels were quantified by qRT-PCR in T-Rex/K-ras cells incubated with or without doxycycline for 72 h. (C) Secretion of EGF, FGF1 and IL-1α in the culture medium of K-ras/On or Off cells, measured by ELISA. The results were normalized by protein contents of the corresponding cell samples. (D) FGFR1, EGFR and K-Ras protein levels were detected by immunoblotting in T-Rex/K-ras/On (72 h) or Off cells. Data are representative of three separate experiments. (E) T-Rex/K-ras cells were incubated with EGF (20 ng/ml), FGF1 (10 ng/ml), or IL-1α (100 ng/ml) for 24 h, and PD-L1 mRNA was quantified by qRT-PCR. (F) T-Rex/K-ras cells were incubated with EGF (20 ng/ml), FGF1 (10 ng/ml) of IL-1α (100 ng/ml) for 48 h. PD-L1 protein was detected by immunoblotting. (G) T-Rex/K-ras cells were incubated with EGF (20 ng/ml), FGF1 (10 ng/ml) or IL-1α (100 ng/ml) for 72 h, and cell surface PD-L1 was analyzed by flow cytometry. Statistical analysis: Data are means ± SEM of three separate experiments; One-way ANOVA followed by Tukey post hoc test for A, C, E, G; Two-tailed unpaired t-test for B. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3: FGFR2 is a direct target of miR-628. (A) TargetScan and miRDB bioinformatics software was used to predict the potential binding site for miR-628-5p binding site in the wild-type and mutant 3′UTR of FGFR2. Luciferase activity of the wt or mut 3′UTR of FGFR2 following transfection with (B) miR-628 mimic or (C) miR-628 inhibitor. **P<0.01 vs. control. miR, microRNA; FGF1, fibroblast growth factor 1; FGFR2, fibroblast growth factor receptor 2; UTR, untranslated region; wt, wild-type; mut, mutant.
Fig 4: Correlation analysis between the serum expression levels of miR-628 and FGF-1 or FGFR2 in patients with PCa. Pearson correlation coefficient analyses were performed to determine the association between the serum expression level of miR-628 and (A) FGF1 or (B) FGFR2. miR, microRNA; FGF1, fibroblast growth factor 1; FGFR2, fibroblast growth factor receptor 2; PCa, prostate cancer.
Fig 5: Expression levels of FGF1 and FGFR2 in patients with PCa. Serum expression levels of (A) FGF1 and (B) FGFR2 in patients with PCa and healthy controls. **P<0.01 vs. control. FGF1, fibroblast growth factor 1; FGFR2, fibroblast growth factor receptor 2; PCa, prostate cancer.
Supplier Page from Abcam for Human FGF1 ELISA Kit