Fig 2: N-linked glycosylation is required for proper BMP/SMADs signaling. (A) Left panel. Representative immunoblots of TFR2, HFE, HJV, and TMPRSS6 proteins in total cells lysate of HuH7 sh-74 and sh-CTR cells. GAPDH is the loading control. Right panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01; Student’s t-test). (B) Left panel. Representative immunoblots of TFR2, HFE, and TMPRSS6 in HuH7 control cells treated with DMSO (vehicle) or increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). GAPDH is the loading control. Right panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation. (** p < 0.01, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, °° p < 0.01 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle, §§ p < 0.01 0.5 ug/mL tunicamycin-treated cells vs. vehicle; * p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle; ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). (C) Upper panel. Representative immunoblots of pSMAD1/5/8 and tSMAD1/5/8 proteins in HuH7 control cells treated with DMSO (vehicle) and increasing concentration of tunicamycin (0.5, 1.0, 1.5 µg/mL). Lower panel. Quantification by densitometric analysis of three separate Western blots with similar results. Data are means ± standard deviation (* p < 0.05, 1.5 μg/mL tunicamycin-treated cells vs. vehicle, ° p < 0.05 of 1.0 µg/mL tunicamycin-treated cells vs. vehicle. ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). (D) Quantification of HAMP gene expression in sh-CTR cells treated with DMSO and tunicamycin (1.5 µg/mL). Data are means ± standard deviation of three independent experiments. (** p < 0.01; Student’s t-test).

Fig 3: Differential regulation of hepcidin antimicrobial peptide (HAMP) and its inhibitor TMPRSS6 during hepatitis C virus (HCV) infection. HAMP and HCV NS3 mRNA expression (a) and hepcidin-secreted peptide levels (b) were measured in triple-cell co-cultures following infection with HCV JFH-1. RNA was isolated from Huh7.5 hepatoma cells and THP-1 macrophages and subjected to RT-qPCR at 24 h intervals post-infection (p.i.). 18S rRNA was used as an internal control. Secreted peptide concentrations were measured by ELISA in pooled supernatants. The mock-infected controls per time interval were arbitrarily set as 100% (represented by the dashed line), and all plotted values are the percentage of these. (c) TMPRSS6 was detected in Huh7.5 whole cell extracts from the same triple-cell co-cultures described in (a,b). Isolated protein was used in immunoblotting analysis, and β-actin was the loading control. The blots were subjected to densitometry and plotted in expression histograms. Values were calculated as a percentage of the respective mock-infected controls (represented by the dashed line). All graphs depict the average expression from at least three individual experiments, with representative accompanying blots. Statistical significance is denoted by asterisks as follows: *, p-value ≤ 0.05; **, p-value ≤ 0.005.