Fig 2: The mRNA and protein expression of representative FGF family members in placentas of STZ induced GDM animal models. (A–F) The mRNA levels of Fgf1, Fgf4, Fgf7, Fgf8, Fgf9, and Fgf21 were analyzed using RT-PCR. (G–I) The protein expression levels of the placental FGF1, FGF4 and FGF21 were detected using ELISA kit. These experiments were performed using fifteen placentas from different mice per group. Data are presented as mean values ±SEM (n = 15). (J) The FGF4 protein expression levels were detected using western blotting (three placentas from different mice per group), and representative images from two independent experiments were shown. (K) Densitometric quantification of western blots shown in (J). Data are presented as mean values ±SEM (n = 6); a value of p < 0.05 was considered to be statistically significant.

Fig 3: The endogenous expression of representative FGF family members and their physiological correlation in human GDM and normal gestational placentas (A–F) The mRNA levels of Fgf1, Fgf4, Fgf7, Fgf8, Fgf9, and Fgf21 in placentas were analyzed using RT-PCR. (G–I) The FGF1, FGF4 and FGF21 expression levels were detected using ELISA analysis. (J-L) The correlation between growth factors (FGF1, FGF4, and FGF21) and blood glucose. Data are presented as mean values ±SEM (n = 18–19); (M,N) Protein levels of FGF4, phosphorylation of AKT (p-AKT), p-AMPK/AMPK, CD68 and TNF-α in human placentas were detected using western blotting. Data are presented as mean values ±SEM (n = 15); a value of p < 0.05 was considered to be statistically significant.

Fig 4: Impact of FGF4 on the IL6/STAT3 axis and macrophage polarization. A The experimental workflow diagram illustrates the construction of FGF4 overexpressing and knockdown TNBC cell lines, co-culture with macrophages, and subsequent analysis procedures. B qRT-PCR analysis of FGF4 mRNA expression levels in MDA-MB-231 cells with FGF4 overexpression or knockdown. C Western blot analysis of STAT3, p-STAT3, and IL-6 expression in macrophages co-cultured with MDA-MB-231 cells under different FGF4 expression conditions. D Quantification of protein expression levels. E Immunofluorescence detection of M1 macrophage polarization markers (iNOS, CD86) in macrophages. F Quantification of iNOS fluorescence in each group. G Quantification of CD86 fluorescence in each group. H Immunofluorescence staining for M2 macrophage polarization markers (Arg1, CD206) in macrophages. I Quantification of CD206 fluorescence in each group. J Quantification of Arg1 fluorescence in each group. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All cell experiments were performed in triplicate

Fig 5: Effects of FGF4 on tumor growth and the immune microenvironment in a TNBC mouse model. A Schematic diagram of the orthotopic tumor model, showing the implantation of FGF4 overexpressing, knockdown, and control breast cancer cells into the mammary fat pads of immunocompetent mice. B Representative images of tumors and tumor weight measurements from each group of mice. C–G In tumor tissues, Immunohistochemistry staining and quantitative analysis of M2 macrophage markers (Arg1, CD206) and M1 macrophage markers (iNOS, CD86). H, I ELISA measurement of immune suppressive cytokine IL-10 levels in tumor tissue. (J-K) Flow cytometry analysis of IFN-γ, TNF-α, and GzmB expression levels in tumor-infiltrating T cells, assessing T cell anti-tumor activity. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each group included 10 mice