Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: After peroxidase blocking, sections were incubated with primary antibodies against FCN3 (#11867-1-AP, 1:200; Proteintech, Wuhan, Hubei, China) or STT3A (#12034-1-AP, 1:200; Proteintech) at 37 °C for 30 min.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Knockdown, Flow Cytometry, Co-Culture Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: After peroxidase blocking, sections were incubated with primary antibodies against FCN3 (#11867-1-AP, 1:200; Proteintech, Wuhan, Hubei, China) or STT3A (#12034-1-AP, 1:200; Proteintech) at 37 °C for 30 min.

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: After peroxidase blocking, sections were incubated with primary antibodies against FCN3 (#11867-1-AP, 1:200; Proteintech, Wuhan, Hubei, China) or STT3A (#12034-1-AP, 1:200; Proteintech) at 37 °C for 30 min.

Techniques: Glycoproteomics, Electrophoretic Mobility Shift Assay, Modification, Knockdown, Transfection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: After peroxidase blocking, sections were incubated with primary antibodies against FCN3 (#11867-1-AP, 1:200; Proteintech, Wuhan, Hubei, China) or STT3A (#12034-1-AP, 1:200; Proteintech) at 37 °C for 30 min.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: After peroxidase blocking, sections were incubated with primary antibodies against FCN3 (#11867-1-AP, 1:200; Proteintech, Wuhan, Hubei, China) or STT3A (#12034-1-AP, 1:200; Proteintech) at 37 °C for 30 min.

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Knockdown, Flow Cytometry, Co-Culture Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Glycoproteomics, Electrophoretic Mobility Shift Assay, Modification, Knockdown, Transfection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: STT3A promoted HCC development by regulating Treg cell activation in vivo. ( A ) Representative tumors of C57BL/6 mice at day 21 post-inoculation of Hepa1-6 cells with sh-STT3A or sh-NC transfection. ( B - C ) Tumor volume and body weight recorded during 21 days post Hepa1-6 cell inoculation demonstrated the effect of sh-STT3A on HCC tumor growth. ( D - F ) qRT-PCR and WB analysis of FOXP3 and CD25 in tumor tissues from HCC mice with sh-STT3A or sh-NC. ( G ) H&E-stained lung sections showing the impact of sh-STT3A on the number of metastatic nodules in HCC mice. ( H - J ) Tumor growth evaluation (images, volume, weight) in mice receiving Hepa1-6 cells with OE-NC, OE-STT3A, or OE-STT3A + DT. ( K - L ) ELISA assessing the influence of OE-STT3A and OE-STT3A + DT on TGF-β1 and IL-10 in HCC mouse tumor tissues. ( M ) Assessment of lung metastasis in HCC mice with OE-NC, OE-STT3A, or OE-STT3A + DT treatment using H&E staining. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. sh-NC/ OE-NC/ OE-STT3A

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, In Vivo, Transfection, Quantitative RT-PCR, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Knockdown, Flow Cytometry, Co-Culture Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Glycoproteomics, Electrophoretic Mobility Shift Assay, Modification, Knockdown, Transfection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Knockdown, Flow Cytometry, Co-Culture Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Glycoproteomics, Electrophoretic Mobility Shift Assay, Modification, Knockdown, Transfection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: STT3A promoted HCC development by regulating Treg cell activation in vivo. ( A ) Representative tumors of C57BL/6 mice at day 21 post-inoculation of Hepa1-6 cells with sh-STT3A or sh-NC transfection. ( B - C ) Tumor volume and body weight recorded during 21 days post Hepa1-6 cell inoculation demonstrated the effect of sh-STT3A on HCC tumor growth. ( D - F ) qRT-PCR and WB analysis of FOXP3 and CD25 in tumor tissues from HCC mice with sh-STT3A or sh-NC. ( G ) H&E-stained lung sections showing the impact of sh-STT3A on the number of metastatic nodules in HCC mice. ( H - J ) Tumor growth evaluation (images, volume, weight) in mice receiving Hepa1-6 cells with OE-NC, OE-STT3A, or OE-STT3A + DT. ( K - L ) ELISA assessing the influence of OE-STT3A and OE-STT3A + DT on TGF-β1 and IL-10 in HCC mouse tumor tissues. ( M ) Assessment of lung metastasis in HCC mice with OE-NC, OE-STT3A, or OE-STT3A + DT treatment using H&E staining. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. sh-NC/ OE-NC/ OE-STT3A

Article Snippet: For detection of secreted FCN3, the Human FCN3 ELISA Kit (#CSB‐E13104h, Cusabio, Wuhan, Hubei, China) was used following the supplier’s instructions.

Techniques: Activation Assay, In Vivo, Transfection, Quantitative RT-PCR, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Knockdown, Flow Cytometry, Co-Culture Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Glycoproteomics, Electrophoretic Mobility Shift Assay, Modification, Knockdown, Transfection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: 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

Article Snippet: For FCN3 staining, HCC‐LM3 and Hep3B cells were similarly processed and probed with FCN3 primary antibody (#11867‐1‐AP, 1:200; Proteintech) overnight at 4 °C, followed by incubation with Cy3‐conjugated goat anti‐rabbit IgG (#A0516, 1:100; Beyotime).

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 1. The expression of FCN3 in HCC tumor and paired adjacent non-tumor tissues. (A) FCN3 mRNA levels in 7 cases/patients by RT-qPCR. Data pre sented are mean ± SEM of three replicate measurements. Statistical signifi cance of the differences is assessed by two-tailed t-test: ***, P < 0.001. (B) FCN3 protein levels in 3 cases by Western blotting assay. GAPDH levels were used to verify equal sample loading. The density of each FCN3 band is plotted as relative FCN3 protein expression. (C) Reduced FCN3 protein levels in tumors as compared with adjacent non-tumor tissues in 4 cases by immunofluorescence staining. The scale bar = 20 μm. The green color represents positive staining for FCN3 protein as indicated by arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Immunofluorescence, Staining

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 2. Confirmation of ectopic FCN3 over-expression. An FCN3 expression vector and matched control vector were transfected into HCC cell lines via lentiviral infection. FCN3 stable over-expression (OE) in Huh7 (A) and SNU398 (B) cells at its mRNA level was confirmed with RT-qPCR. Data presented are mean ± SEM of three replicate measurements. Statistical significance of the differences is assessed by two-tailed t-test: ***, P < 0.001. FCN3 stable over-expression was confirmed at its protein level with Western blotting (C) and immnocytochemistry (D). The scale bar = 10 μm. The expression of FCN3 in a paired adjacent (Adj) non-tumor and tumor tissues of an HCC patient is shown for comparison between FCN3 levels in Adj tissue and FCN3 OE cells in Panel C. Tublin protein levels were used to verify equal sample loading. The density of each FCN3 band is plotted as relative FCN3 protein expression.

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Transfection, Infection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Comparison

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 3. FCN3 over-expression inhibited the growth and migration of HCC cells. Anchorage-dependent growth of the paired cell lines was assessed with confluence assay (A, C and E (the scale bar = 1000 μm)) and MTT assay (B and D). Data presented are mean ± SEM of four replicate measurements. Statistical significance of the differences is assessed by two-way ANOVA test: ***, P < 0.001. FCN3 over-expression inhibited the anchorage-independent growth of the paired HCC cell lines in soft agar assay (F), and their migration in the transwell assay (G The scale bar = 200 μm). Data presented are mean ± SEM of three replicate measurements for Migration and Soft agar assay. Statistical significance of the differences is assessed by two-tailed t-test: **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Over Expression, Migration, MTT Assay, Soft Agar Assay, Transwell Assay, Two Tailed Test

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 4. Over-expression of FCN3 induced cell apoptosis. The paired SNU398 and Huh7 cells were stained with propidium iodide (PI) and AnnexinV-FITC and AnnexinV positive apoptotic cells were scored with flow cytometry (A, B). Apoptotic cells with cytoplasmic DNA were also detected with an apoptosis ELISA assay (C). Data presented are mean ± SEM of three replicate measurements from the ELISA assay. Statistical significance of the differences is assessed by two-tailed t-test: ***, P < 0.001.

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Over Expression, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 5. Over-expression of FCN3 inhibited xenograft tumor growth in vivo. Growth curve of tumors formed by Huh7 (A) and SNU398 (B) Ctl and matched FCN3 OE cells in nude mice. The tumor volume was calculated using the formula: v = length × width × width / 2. The survival curves were generated by defining the mice with tumor volumes <2 cm3 as “live” and larger than 2 cm3 as “dead” (C, D). Statistical significance of the differences of survival curve is assessed by two-way ANOVA test: **, P < 0.01. ***, P < 0.001. FCN3 protein expression in SNU398 Ctl and matched FCN3 OE xenograft tumors was detected with immunofluores cence staining (E). The scale bar = 10 μm.

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Over Expression, In Vivo, Generated, Expressing, Staining

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 7. Complement activation contributed to FCN3-induced growth inhibition. (A) Immunocytochemical staining of SNU398 and Huh7 control and FCN3 OE cells treated with 10 % Hi-HPS or HPS for 30 min with an anti-C5b-9 antibody. DAPI-stained nuclear DNA is shown as blue color. (B) Immunocytochemical staining of SNU398 and Huh7 parental cells treated for 30 min with 10 % FBS, Hi-HPS, or HPS in the absence or presence of rFCN3 at 200 nM based on its monomer molecular weight of 33 kD. (C) SNU398 cells were plated in a 96-well plate at 20,000 cells/well in the culture medium, which was replaced next day with 10 % Hi-HPS or HPS in RPMI1640 medium with or without active or heat-inactivated rFCN3 (Hi, 50 ◦C for 30 min). After 36 h treatment, viable cells were quantified with MTT assay. Data presented are mean ± SEM of four replicate measurements. P values were obtained with one-way ANOVA followed with Tukey’s multiple comparison test. (D) SNU398 cells were cultured in the regular 10 % FBS RPMI 1640 medium without or with 5 μg/ml mitomycin c (Mc) as positive control for cell death or in 10 % Hi- HPS or HPS RPMI 1640 medium with or without rFCN3 for 16 h and then stained with PI. Total and PI positive cells in five high-power fields were counted to calculate mean ± SEM percent PI positive cells. P values were obtained with one-way ANOVA followed with Tukey’s multiple comparison test. Representative PI- stained cell images of each treatment are shown. (E) SNU398 control and FCN3 OE cells were treated with the anti-C6 blocking antibody at 20 μg/ml over a 6-day period. The cell confluence in each well was plotted as mean ± SEM from triplicate wells. Representative images of the cell confluence at Day 6 are shown. Statistical significance due to anti-C6 antibody treatment in control or FCN3 OE cells respectively were assessed with two-way repeated measures ANOVA tests.*, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001. The scale bar = 10 μm in Panel A & B, =100 μm in Panel D, =1000 μm in Panel E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: Activation Assay, Inhibition, Staining, Control, Molecular Weight, MTT Assay, Comparison, Cell Culture, Positive Control, Blocking Assay

Journal: Life sciences

Article Title: Ficolin-3 induces apoptosis and suppresses malignant property of hepatocellular carcinoma cells via the complement pathway.

doi: 10.1016/j.lfs.2024.123103

Figure Lengend Snippet: Fig. 8. Schematic diagram showing that FCN3 induces apoptosis and inhibits viability of HCC cell via the complement lectin pathway which can be blocked by anti- C6 antibody.

Article Snippet: After blocking, the cells were incubated with 250 μl anti-FCN3 primary antibody from rabbit (1:200 dilution in 5 % goat serum, LS Biologicals, Seattle, WA, Cat.#: LS-C166110), anti-C3b primary antibody from rabbit (1:10,000 dilution, Proteintech, Cat: No. 21337–1-AP) or anti-C5b-9 primary antibody from rabbit (1:1,000 dilution, Abcam, Cat.#: 55811) at 4 ◦C overnight and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:3,000 dilution in PBS, Abcam, Cambridge, MA, Cat.#: ab150077) at room temperature for 1 h. Washing step of 5 min for 3 times using PBS was applied after primary and secondary antibody incubation.

Techniques: