Fig 1: Enhanced N-glycosylation of FCN3 in HCC. (A) Protein-protein interaction network obtained from BioGRID Database exhibited the connection between FCN3 and glycosylation-related proteins. (B) WB assay confirmed FCN3 band shift in HCC cell lysates, showing FCN3 galactosylated modification. (C) WB analysis of HCC cell lysates treated with PNGase F or O-Glycanase was performed to verify that FCN3 glycosylation was N-linked rather than O-linked. (D) FCN3 band shift in Hep3B cells treated with TM or DMSO was analyzed by WB. (E) WB analysis of FCN3 protein was performed in FCN3-knockdown HCC cells transfected with either Vector, FCN3-WT, or FCN3-N189Q. (F-G) IF staining and ELISA quantification showed the effects of the Asn189 mutation on FCN3 intracellular levels and its secretion into the culture supernatant, respectively
Fig 2: FCN3 regulated Wnt/β-catenin signaling to influence Treg activation. (A-B) qRT-PCR and WB analysis of APC and β-catenin in HCC tumor and adjacent non-tumor tissues. (C) Clinical sample IF staining showing β-catenin expression and localization with DAPI nuclear counterstain. (D-E) The levels of APC and β-catenin in HCC cell lines were assessed by qRT-PCR and WB. (F-G) qRT-PCR and WB evaluation of APC and β-catenin in Hep3B cells treated with OE-NC, OE-FCN3, OE-FCN3 + DMSO, or OE-FCN3 + LY2090314 (β-catenin activator). (H) The levels of TGF-β1 and IL-10 in cell culture supernatant of each group were examined by ELISA. (I-M) Wound healing, Transwell and flow cytometry assays evaluating the effect of OE-FCN3 and OE-FCN3 + LY2090314 on cell migration, invasion and apoptosis of Hep3B cells. (N) Flow cytometry was performed to analyze the effects of OE-FCN3 and OE-FCN3 + LY2090314 treatment in Hep3B cells on the proportion of CD4⁺CD25⁺FOXP3+ Treg cells in Hep3B-PBMC co-cultures. ***p < 0.001, **p < 0.01, *p < 0.05 vs. N/ THLE-2/ OE-NC/ OE-FCN3 + DMSO
Fig 3: STT3A involved in HCC progression via promoting FCN3 N-glycosylation. (A) Bioinformatics analysis revealing STT3A expression in normal and HCC liver tissues. (B-D) qRT-PCR, WB and IHC assays were performed to assess STT3A levels in HCC tumor and adjacent non-tumor tissues. (E-F) The mRNA and protein levels of STT3A in normal hepatocyte and HCC cell lines were analyzed by qRT-PCR and WB. (G) Correlation analysis between STT3A protein expression and FCN3 protein expression in tumor tissues from HCC patients. (H) Co-IP assay verified STT3A-FCN3 interaction. (I) WB analysis of FCN3 glycosylation in Hep3B cells transfected with sh-STT3A or sh-NC. (J-K) The effects of sh-STT3A on the expression of APC and β-catenin in Hep3B cells were examined by qRT-PCR and WB. (L) ELISA showing TGF-β1 and IL-10 levels in Hep3B cell supernatants following sh-STT3A or sh-NC treatment. (M-Q) Wound healing, Transwell and flow cytometry assays were conducted to evaluate the effects of sh-STT3A on Hep3B cell migration, invasion and apoptosis. (R) The influence of sh-STT3A on the proportion of CD4⁺CD25⁺FOXP3+ Treg cells in Hep3B-PBMC co-cultures was assessed by flow cytometry. ***p < 0.001, **p < 0.01, *p < 0.05 vs. N/ THLE-2/ sh-NC
Fig 4: N-glycosylated FCN3 influenced Wnt/β-catenin signaling and Treg activation. (A) qRT-PCR analysis of APC and β-catenin mRNA levels in Hep3B cells transfected with FCN3-WT or FCN3-N189Q. (B) WB analysis showing the levels of APC, β-catenin, and p-β-catenin. (C) Subcellular localization of β-catenin was analyzed by WB. (D) ELISA was conducted to assess the levels of TGF-β1 and IL-10 in cell culture supernatant of Hep3B cells transfected with FCN3-WT or FCN3-N189Q. (E-K) The effects of FCN3 glycosylation on cell viability, migration, invasion and apoptosis in Hep3B cells were respectively evaluated by CCK-8, wound healing, Transwell and flow cytometry assays. (L) Flow cytometry demonstrating the impact of FCN3 glycosylation on the proportion of CD4⁺CD25⁺FOXP3+ Treg cells in Hep3B-PBMC co-cultures. ***p < 0.001, **p < 0.01, *p < 0.05 vs. FCN3-WT
Fig 5: Low FCN3 expression in HCC affected Treg cell activation. (A) Kaplan-Meier survival analysis showing the correlation between FCN3 levels and overall survival of HCC patients. (B-D) qRT-PCR, WB and IHC analysis of FCN3 expression in tumor (T) and adjacent non-tumor (N) tissues from HCC patients. (E-F) The levels of FCN3 in normal hepatocyte THLE-2 and HCC cell lines (HepG2, Hep3B, HCC-LM3) were assessed by qRT-PCR and WB. (G-H) qRT-PCR and WB analysis demonstrating the mRNA and protein levels of Treg cell markers FOXP3 and CD25 in clinical samples. (I) ELISA quantification of TGF-β1 and IL-10 in HCC tumor and adjacent non-tumor tissues. (J-K) The levels of FOXP3 and CD25 in PBMCs co-cultured with FCN3-knockdown or FCN3-overexpressing HCC cells were evaluated by qRT-PCR and WB. (L) Flow cytometry analysis of CD4⁺CD25⁺FOXP3+ Treg cell proportion in PBMCs within the co-culture systems. ***p < 0.001, **p < 0.01, *p < 0.05 vs. N/ THLE-2/ sh-NC/ OE-NC
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