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 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 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:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 1 The reduced FCN3 level in HCC are associated with ferroptosis resistance (A) The complement signaling pathway associated with prognosis (green represents unfavorable, purple represents favorable) in various tumors was analyzed by Kaplan–Meier analysis based on TCGA database. (KIRP: Kidney renal papillary cell carcinoma, CESC: Cervical squamous cell carcinoma, DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, PAAD: Pancreatic adenocarcinoma, CHOL: Cholangio carcinoma, OV: Ovarian serous cystadeno carcinoma, BLCA: Bladder Urothelial Carcinoma, COAD: Colon adenocarcinoma, STAD: Stomach adenocarcinoma, LUSC: Lung squamous cell carcinoma, GBM: Glioblastoma multiforme, KIRC: Kidney renal clear cell carcinoma, KICH: Kidney Chromophobe, UVM: Uveal Melanoma). (B) mRNA expression of complement-related genes in normal human liver tissues (N) and HCC tissues (T) in published data from TCGA database. (C) mRNA expression of FCN3 in 15 groups of HCC (T), PVTT (P) and paired adjacent nontumor (N) tissues. log2(T/N) value < 0 indicated that FCN3 expression was downregulated, while log2(T/N) value > 0 indicated that FCN3 expression was upregulated in HCC and PVTT samples. (D-E) Immunoblot (D) and quantification (E) of FCN3 in 18 pairs of HCC (T) and adjacent nontumor (N) tissues. GAPDH was used as a loading control. FCN3 exhibited two bands in human samples (two transcripts). (F) Kalpan–Meier analysis of disease-free survival of tissue microarray (TMA) data containing 134 patients. (G-H) mRNA expression of PTGS2 (G) and CBS (H) in normal human liver tissues (N) and HCC tissues (T) in data from TCGA database. (I) Correlation analysis of PTGS2 and FCN3 expressions based on TCGA database. (J) Representative images of FCN3-overexpressed YY-8103 and MHCC97-H cells treated with erastin. Scale bar, 100 μm. (K-L) Quantification of trypan blue staining for death cell in FCN3-overexpressed YY-8103 (K) and MHCC97-H (L) cells treated with erastin in (J). (M) FCN3-overexpressed YY-8103 cells were treated with erastin (10 µM) and specific cell death inhibitors. Scale bar, 100 μm Data are from one representative experiment of three independent experiments (C-D and J-M). Data are presented as mean ± SD. Significance was as sessed by Mann-Whitney U test (B, E, G, H), Log-rank test (F), Spearman correlation (I), Student’s t test (K, L). **p < 0.01, ***p < 0.001 compared with the control group

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Expressing, Western Blot, Control, Microarray, Staining, MANN-WHITNEY

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 2 Intracellular FCN3 inhibits the migration and survival of HCC cells by promoting ferroptosis (A-B) Representative images (A) and quantification (B) of migrating MHCC97-H cells after treatment with 300 ng/mL FCN3 recombinant protein. Scale bar, 100 μm. (C-D) Crystal violet (C) and CCK8 assay (D) in FCN3 recombinant protein treated MHCC97-H cells, the treatment medium was refreshed every three days, FCN3 recombinant protein: 300 ng/mL. (E) Representative images of the crystal violet assay in nsFCN3-overexpressed YY-8103 and Huh7 cells. (F) CCK8 assay in nsFCN3-overexpressed MHCC97-H cells. (G) Representative images of migrating MHCC97-H and YY-8103 cells that cultured in trans-well plates. Scale bar, 100 μm. (H) Quantification of the average number of migrating MHCC97-H and YY-8103 cells in (G). (I) Representative images of nsFCN3- overexpressed MHCC97-H cells treated with erastin. Scale bar, 100 μm. (J) Quantification of trypan blue staining for death cell in nsFCN3-overexpressed MHCC97-H cells treated with erastin. (K-L) Viability of FCN3- and nsFCN3-overexpressed MHCC97-H cells treated with indicated concentrations of erastin (K) or FINO2 (L) for 24 h. (M) Representative images of nsFCN3-overexpressed YY-8103 cells treated with FINO2 (10 µM) and Ferr-1 (2 µM). Scale bar, 100 μm. (N-O) Representative images (N) and quantification (O) of migrating YY-8103 cells treated with Ferr-1 (2 µM). Scale bar, 100 μm Data are presented as mean ± SD and are from one representative experiment of three independent experiments. Significance was assessed by Student’s t test (B, H, J, O). **p < 0.01, ***, ###p < 0.001 compared with the control group. ns, not significant

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Migration, Recombinant, CCK-8 Assay, Crystal Violet Assay, Cell Culture, Staining, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 3 FCN3 inhibits the development of HCC in vivo (A) Immunoblot of FCN3 in the liver of FCN3LKI and control mice. Gapdh was used as a loading control. (B) Tumor incidence from FCN3LKI and control mice inducted for 28 and 32 weeks. (C) Quantification of total surface tumors number from FCN3LKI and control mice inducted for 32 weeks. (D) Representative images of the livers and tumors from FCN3LKI and control mice inducted for 28 and 32 weeks. Scale bar, 200 μm & 40 μm. (E) Ratios of liver weight to body weight at week 32. (F) Representative images of Ptgs2 staining in mouse liver. Scale bar, 20 μm. (G) Quantification of Ptgs2 staining in (F). (H) Representa tive image of xenografts for nsFCN3-overexpressed and control MHCC97-H cells at day 36 (n = 6 mice per group). (I) Weights of xenograft tumors in (H) at day 36 (n = 12 per group). (J) A subcutaneous tumor volume curve from (H) (n = 6 mice per group). (K) Representative images of FCN3 and ACSL4 IHC staining in xenograft tumors. Scale bar, 40 μm. (L) Immunoblots of ACSL4 and NRF2 in xenograft tumors. β-Tubulin was used as a loading control. (M) mRNA levels of ferroptosis-related genes in xenograft tumors. (N) Representative images of Ki67 IHC staining in xenograft tumors. Scale bar, 20 μm. (O) Immunoblots of EMT-related proteins in xenograft tumors. β-Actin was used as a loading control Data are presented as mean ± SD. Significance was assessed by Mann-Whitney U test (C), Student’s t test (E, G, I, M). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: In Vivo, Western Blot, Control, Staining, Immunohistochemistry, MANN-WHITNEY

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 4 FCN3 promotes ferroptosis in HCC cells by reducing MUFA levels and enhancing lipid oxidation (A) Immunoblot of GPX4 in nsFCN3-overexpressed YY-8103 cells treated with 10 µM erastin for 24 h. GAPDH was used as a loading control. (B) mRNA levels of SLC7A11 and SLC3A2 in nsFCN3-overexpressed MHCC97-H cells and control cells. (C) mRNA levels of iron metabolism-related genes in xenograft tumors derived from Fig. 3H. (D) Representative images of FTH1 IHC staining in xenograft tumors derived from Fig. 3H. Scale bar, 40 μm. (E) The diagram of GO enrichment based on the DEGs (|log2 Fold Change|>1, p value < 0.05) in nsFCN3-overexpressed and control MHCC97-H cells analyzed with RNA- seq (n = 3). (F-G) mRNA levels of lipid peroxidation-related genes in nsFCN3-overexpressed MHCC97-H (F) cells and YY-8103 (G) cells. (H) The content of MDA in nsFCN3-overexpressed and control MHCC97-H cells (n = 5). (I-J) Representative images (I) and quantification (J) of BODIPY-C11 stained nsFCN3- overexpressed MHCC97-H and YY-8103 cells treated with 10 µM erastin for 12 h. A shift in green-to-red ratio indicates lipid oxidation, Scale bar, 40 μm. (K) Representative images of nsFCN3-overexpressed YY-8103 cells treated with FINO2 (10 µM) and NAC (1 mM). Scale bar, 100 μm. (L) Heatmap analysis of MUFA in tumor tissues (T) compared to paired normal adjacent tissue (N). Red indicated increase, and blue indicated decrease. -2 ~ 2 indicated the Fold Change. (M) Volcano plot of metabolites in nsFCN3-overexpressed and control MHCC97-H cells. LC-MS-based nontargeted metabolomic analysis, and the data was corrected by total peak area. (N-O) Fatty acid levels in YY-8103 (N) and MHCC97-H (O) cells overexpressing nsFCN3 Data are from one representative experiment of three independent experiments (F-G). Data are presented as mean ± SD. Significance was assessed by Student’s t test (B, C, F, G, H, J), Mann-Whitney U test (N, O). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group. ns, not significant

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Western Blot, Control, Derivative Assay, Immunohistochemistry, RNA Sequencing, Staining, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 5 FCN3 inhibits MUFA synthesis by downregulating SREBP1c expression (A) Enrichment of lipid metabolism related biological processes based on the GO enrichment in Fig. 4E. (B-C) mRNA levels of lipogenic genes in nsFCN3- overexpressed YY-8103 (B) and MHCC97-H (C) cells. (D) mRNA levels of lipogenic genes in FCN3-knockdown and control Huh7 cells. (E-F) Immunoblots of lipogenic proteins in nsFCN3-overexpressed YY-8103 (E) and MHCC97-H (F) cells. GAPDH was used as a loading control. (G) Immunoblots of lipogenic proteins in FCN3-knockdown and control Huh7 cells. GAPDH was used as a loading control. (H-I) Immunoblots (H) and quantification (I) of lipogenic proteins in the liver tissues of FCN3LKI and control mice. GAPDH was used as a loading control. (J) mRNA levels of lipogenic genes in the liver tissues of FCN3LKI and control mice. (K-M) Correlation analysis of ACC (K), FASN (L), SCD (M) and FCN3 expressions based on TCGA database. (N-P) mRNA levels of SREBP1c (N), SCD (O) and ACC (P) in 15 paired adjacent and tumor tissues. Data are from one representative experiment of three independent experiments (B-G). Data are presented as mean ± SD. Significance was assessed by Student’s t test (B, C, I, J), one-way ANOVA (D), Spearman correlation (K, L, M), paired Student’s t test (N, P), Wilcoxon matched-pairs signed rank test (O). *,

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Expressing, Knockdown, Control, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 6 FCN3 suppresses HCC by inhibiting the AKT/SREBP axis (A) Immunoblots of DNL-related proteins in nsFCN3-overexpressed YY-8103 cells treated with T0901317 (10 µM) for 24 h. GAPDH was used as a loading control. (B) mRNA levels of lipogenic genes in nsFCN3-overexpressed and control YY-8103 cells treated with T0901317 (10 µM) for 24 h. (C) TG contents in nsFCN3-overexpressed YY-8103 cells treated with T0901317 (10 µM) and palmitic acid (PA, 100 µM). (D) Representative images of indicated cells that cultured in trans-well plates. Scale bar, 100 μm. (E) Quantification of the average number of migrating YY-8103 and MHCC97-H cells in (D). (F) mRNA levels of lipogenic genes in nsFCN3-overexpressed YY-8103 cells treated with AKTi (10 µM) for 24 h. (G) Immunoblots of lipogenic proteins and p-AKT in nsFCN3-overexpressed YY-8103 and MHCC97-H cells treated with AKTi (10 µM) for 24 h. GAPDH was used as a loading control. (H) Representative images of nsFCN3-overexpressed YY-8103 cells treated with FINO2 (5 µM) and AKTi (10 µM). Scale bar, 100 μm. (I) Quantification of trypan blue staining for death cell in (H). (J) Representative images of indicated cells that cultured in trans-well plates. Scale bar, 100 μm. (K) Quantification of the average number of migrating YY-8103 cells in (J) Data are presented as mean ± SD and are from one representative experiment of three independent experiments. Significance was assessed by Student’s t test (B, C, E, F, I, K). *, #p < 0.05, **, ##p < 0.01, ***, ###p < 0.001 compared with the control group. ns, not significant

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Western Blot, Control, Cell Culture, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 7 FCN3 inhibits IR activation by promoting the dephosphorylation of IR-β and suppressing pro-IR cleavage (A) Immunoblots of p-IR-β and IR-β in nsFCN3-overexpressed MHCC97-H and YY-8103 cells. GAPDH was used as a loading control. (B-C) Quantitative analysis of p-IR-β/IR-β ratio in nsFCN3-overexpressed MHCC97-H (B) and YY-8103 (C) cells. (D-E) Immunoblots (D) and quantification (E) of p-IR-β and IR-β in FCN3-overexpressed YY-8103 cells. GAPDH was used as a loading control. (F-G) Immunoblots of FCN3 and HA-tagged IR after immunoprecipi tation of HA-tagged IR from HEK-293T cells that transfected with FCN3 (F) or nsFCN3 (G) and IR-HA. (H-I) Immunoblots of IR-β, PTP1B and FCN3 after immunoprecipitation of Flag-tagged FCN3 from HEK-293T cells that transfected with FCN3-Flag (H) or nsFCN3-Flag (I). (J) Immunoblots of PTP1B, FCN3 and HA-tagged IR after immunoprecipitation of HA-tagged IR from indicated HEK-293T cells. (K) mRNA level of IR in nsFCN3-overexpressed YY-8103 cells. (L-M) Immunoblots (L) and quantification (M) of pro-IR and IR-β in nsFCN3-overexpressed YY-8103 cells. GAPDH was used as a loading control. (N-O) Im munoblots of pro-IR and FCN3 after immunoprecipitation of Flag-tagged FCN3 from HEK-293T cells that transfected with FCN3-Flag (N) or nsFCN3-Flag (O). (P) Immunoblots of Furin, FCN3 and HA-tagged pro-IR after immunoprecipitation of HA-tagged IR from indicated HEK-293T cells. (Q) Immunoblots of indicated proteins in HEK-293T cells. GAPDH was used as a loading control Data are from one representative experiment of three independent experiments (A-E and K-M). Data are presented as mean ± SD. Significance was as sessed by Student’s t test (B, C, E, K, M). **p < 0.01, ***p < 0.001 compared with the control group. ns, not significant

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Activation Assay, De-Phosphorylation Assay, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

doi: 10.1186/s13046-024-03047-2

Figure Lengend Snippet: Fig. 8 Schematic summary displays that FCN3 deficiency in HCC causes the overactivation of IR-β and AKT, which further promotes the expression of the downstream transcription factor SREBP1c. Then, the expression of DNL and lipid desaturation related genes including ACC1, FASN and SCD1 are up regulated, resulting in increased intracellular MUFA, which promotes a ferroptosis-resistant cell state. In contrast, overexpression of FCN3 enhances lipid peroxidation through inhibiting IR-AKT-DNL axis and thereby sensitizes HCC cells to ferroptosis

Article Snippet: The antibodies were listed with the dilution, catalogue number, and supplier: FCN3, 1:1000, AF2367, R&D Systems; GPX4, 1:1000, 67763-1-lg, Proteintech; SREBP1c, 1:1000, 14088-1-AP, Proteintech; FASN, 1:1000, 10624-2-AP, Proteintech; SCD1, 1:1000, ab236868, Abcam; E-Cadherin, 1:1000, #3195, CST; N-Cadherin, 1:1000, #13116, CST; Slug, 1:1000, #9585, CST; Vimentin, 1:1000, 10366-1-AP, Proteintech; ACC, 1:1000, #3676, CST; p-AKT (S473), 1:1000, #4060, CST; AKT, 1:1000, #4691, CST; IR-β, 1:1000, #23413, CST; p-IGF-IR, 1:1000, #3024, CST; ACSL4, 1:1000, 22401-1-AP, Proteintech; NRF2, 1:1000, A0674, ABclonal; PTP1B, 1:1000, 11334-1-AP, Proteintech; Furin, 1:1000, 18413-1-AP, Proteintech; phosphatase and tensin homolog (PTEN), 1:1000, #9188, CST; protein phosphatase 2 (PP2A) A subunit, 1:1000, #2041, CST; PP2A C subunit, 1:1000, #2259, CST; HA, 1:2000, M2003, Abmart; β-Tubulin, 1:3000, AP0064, Bioworld; GAPDH, 1:3000, A19056, ABclonal; β-Actin, 1:3000, #3700, CST; Hsp90, 1:1000, #4874, CST; Apoptosis antibody sampler kit, #9915, CST; HRP Goat Anti-Rabbit IgG (H + L), 1:50000, E030120, Earthox; HRP Goat AntiMouse IgG (H + L), 1:50000, E030110, Earthox; HRP Donkey Anti-Goat IgG (H + L), 1:5000, AS031, ABclonal.

Techniques: Expressing, Over Expression