Figure 4.
Figure 4. The effect of FGF6 nonsynonymous variants on hepcidin expression and intracellular iron concentration. (A) The effect of FGF-6 active protein treatment on mRNA expression of several iron metabolism genes in HepG2 liver hepatocellular carcinoma cell culture media compared with control. Protein concentration was 250 ng/mL and the incubation time was 24 hours with 10 μM FAC and 500 μM ascorbate in the cell culture media. HAMP encodes for hepcidin. HDAC2 encodes for histone deacetylase 2. HMOX1 encodes for heme oxygenase 1. TFRC encodes for transferrin receptor 1 and HEPH encodes hephaestin. mRNA expression was quantified relative to GAPDH expression. Treatment with PBS served as control. A Student t test was used test for pairwise differences between sets of observations. *P < .05. Results are the mean ± standard deviation (SD) of 3 observations in a single experiment. (B) Iron-metabolism gene expression changes with FGF6 mRNA transfection in the HepG2 cell culture media after 24 hours. Vector without FGF6 served as control. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment. (C-D) Iron-metabolism gene-expression changes after the transfection by FGF6 mRNA into various cell types with WT and the identified variants E172X (M1), D174V (M2), and R188Q (M3). Cell lines: HepG2 are liver hepatocellular carcinoma cells, HCT116 are ileocecal colorectal adenocarcinoma cells, and HFF-1 are human normal skin fibroblasts. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment. (E-F) Total intracellular iron-concentration changes after the transfection with FGF6 mRNA into 3 cell types with WT and the identified M1, M2, and M3 variants in the presence of FAC for 48 hours. A Student t test was used to test for pairwise differences between sets of observations. *P < .05; **P < .01. Results are the mean ± SD of 3 observations in a single experiment. (G-H) Ferritin protein level changes after the transfection by FGF6 mRNA into the 3 cell types with WT and the identified M1, M2, and M3 variants in the presence of FAC for 48 hours. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment.

The effect of FGF6 nonsynonymous variants on hepcidin expression and intracellular iron concentration. (A) The effect of FGF-6 active protein treatment on mRNA expression of several iron metabolism genes in HepG2 liver hepatocellular carcinoma cell culture media compared with control. Protein concentration was 250 ng/mL and the incubation time was 24 hours with 10 μM FAC and 500 μM ascorbate in the cell culture media. HAMP encodes for hepcidin. HDAC2 encodes for histone deacetylase 2. HMOX1 encodes for heme oxygenase 1. TFRC encodes for transferrin receptor 1 and HEPH encodes hephaestin. mRNA expression was quantified relative to GAPDH expression. Treatment with PBS served as control. A Student t test was used test for pairwise differences between sets of observations. *P < .05. Results are the mean ± standard deviation (SD) of 3 observations in a single experiment. (B) Iron-metabolism gene expression changes with FGF6 mRNA transfection in the HepG2 cell culture media after 24 hours. Vector without FGF6 served as control. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment. (C-D) Iron-metabolism gene-expression changes after the transfection by FGF6 mRNA into various cell types with WT and the identified variants E172X (M1), D174V (M2), and R188Q (M3). Cell lines: HepG2 are liver hepatocellular carcinoma cells, HCT116 are ileocecal colorectal adenocarcinoma cells, and HFF-1 are human normal skin fibroblasts. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment. (E-F) Total intracellular iron-concentration changes after the transfection with FGF6 mRNA into 3 cell types with WT and the identified M1, M2, and M3 variants in the presence of FAC for 48 hours. A Student t test was used to test for pairwise differences between sets of observations. *P < .05; **P < .01. Results are the mean ± SD of 3 observations in a single experiment. (G-H) Ferritin protein level changes after the transfection by FGF6 mRNA into the 3 cell types with WT and the identified M1, M2, and M3 variants in the presence of FAC for 48 hours. A Student t test was used to test for pairwise differences between sets of observations. *P < .05. Results are the mean ± SD of 3 observations in a single experiment.

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