reporter constructs p2  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Tripartite motif 8 promotes the progression of hepatocellular carcinoma via mediating ubiquitination of HNF1α

doi: 10.1038/s41419-024-06819-y

Figure Lengend Snippet: A Mass spectrometry analysis revealed the potential interaction between HNF1α and TRIM8. B , C Co-IP assays were performed to detect the interaction between exogenous HNF1α and TRIM8 in HEK293T cells transfected with the plasmids expressing V5-HNF1α and Flag-TRIM8. D In situ proximity ligation assay was performed to detect the interaction between HNF1α and TRIM8 in Huh7 cells. Scale bars = 50 μm. E GSEA analysis of our acquired RNA sequencing data showed an enrichment of HNF1α target genes in the TRIM8-KO groups. Gene correlation analysis between TRIM8 and HNF1α target genes including ALDOB ( F ), APOC3 ( G ) and TTR ( H ) based on TCGA-LIHC database. Luciferase reporter assays were performed to evaluate the activity of the HNF1α promoter in Huh7 cells transfected with the plasmid overexpressing TRIM8 ( I ) or siTRIM8 ( J ). Relative mRNA levels of HNF1α target genes in MHCC-L cells infected with control lentivirus or lenti-TRIM8 ( K ) and MHCC-L cells transfected with control siRNA or siTRIM8 ( L ). M IPA analysis showed the ingenuity canonical pathways of DEGs which were upregulated both in HNF1α-overexpressing cells and in TRIM8-knockout cells. N IPA analysis showed the diseases and bio functions of DEGs which were upregulated both in HNF1α-overexpressing cells and in TRIM8-knockout cells. I – L Experiments were performed in triplicate and data are presented as means ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001 using two- tailed Student’s t tests.

Article Snippet: Cells transfected with HNF1α reporter plasmid pGL3-HNF4a-P2 and the control pRL-SV40 vector (E2261, Promega, USA) together with the plasmid overexpressing TRIM8 or the control plasmid were plated in 96-well plates.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Expressing, In Situ, Proximity Ligation Assay, RNA Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Infection, Control, Knock-Out, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Tripartite motif 8 promotes the progression of hepatocellular carcinoma via mediating ubiquitination of HNF1α

doi: 10.1038/s41419-024-06819-y

Figure Lengend Snippet: The expressions of HNF1α protein were detected in the Huh7 cells transfected with the plasmid overexpressing TRIM8 (Flag-TRIM8) and the control vector ( A ) or the Huh7 cells transfected with siTRIM8 and siNC ( B ). C Huh7 cells were infected with control or Flag-TRIM8 plasmids and then treated with cycloheximide (CHX; 20 μg/mL) for 0 h, 2 h, 4 h, and 6 h. WB analysis was performed to evaluate HNF1α protein levels. D Semi-quantification analysis of HNF1α protein levels after TRIM8 overexpression based on CHX-treated assay. E WB analysis of HNF1α in Huh7 cells after treatment with MG132 (20 µM) for 0 h, 1 h, 2 h, and 3 h. F Huh7 cells were infected with siNC or siTRIM8 and then treated with CHX (20 μg/mL) for 0 h, 2 h, 4 h, and 6 h, and WB was performed to evaluate HNF1α protein levels. G Semi-quantification analysis of HNF1α protein levels after TRIM8-knockdown based on CHX-treated assay. H MG132 (20 µM) was applied to Huh7 cells transfected with V5-HNF1α, HA-Ub, TRIM8 plasmids or control plasmids, and 8 h later, ubiquitination assays were performed to determine the ubiquitination levels of HNF1α. I Ubiquitination assays determined the ubiquitination of endogenous HNF1α in Huh7 cells transfected with plasmid overexpressing TRIM8 or the control plasmid. J The ubiquitination levels of HNF1α after TRIM8 overexpression was examined in Huh7 cells co-transfected with wild-type (WT) and mutated ubiquitin (K48O, K48R, K63R) plasmids. Experiments were performed in triplicate and data are presented as means ± SEM. ns no significance, * P < 0.05 and ** P < 0.01 using two- tailed Student’s t tests.

Article Snippet: Cells transfected with HNF1α reporter plasmid pGL3-HNF4a-P2 and the control pRL-SV40 vector (E2261, Promega, USA) together with the plasmid overexpressing TRIM8 or the control plasmid were plated in 96-well plates.

Techniques: Transfection, Plasmid Preparation, Control, Infection, Over Expression, Knockdown, Ubiquitin Proteomics, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Tripartite motif 8 promotes the progression of hepatocellular carcinoma via mediating ubiquitination of HNF1α

doi: 10.1038/s41419-024-06819-y

Figure Lengend Snippet: A Schematic illustration of tandem ubiquitin-binding entity (TUBE) pull-down assay. B Huh7 cells transfected with V5-HNF1α or V5-HNF1α-K197R plasmids were treated with CHX (20 µg/ml) for 0, 2, 4, and 6 h, and the expression of HNF1α was analyzed by Western blotting (WB) assays. C Semi-quantification analysis of HNF1α protein levels in the indicated groups. D Ubiquitination assays were performed to determine the ubiquitination levels of HNF1α in Huh7 cells transfected with V5-HNF1α or V5-HNF1α-K197R and HA-Ub plasmids together with Flag-TRIM8 plasmids or negative control plasmids. E WB analysis of HNF1α protein levels in Huh7 cells transfected with HNF1α or HNF1α-K197R together with TRIM8 or its control plasmids. F Luciferase reporter assays were performed to evaluate the activity of the HNF1α promoter in Huh7 cells transfected with the indicated plasmids. G Relative mRNA levels of HNF1α target genes in MHCC-L cells infected with control lentivirus or lenti-TRIM8 together with lentivirus expressing HNF1α or HNF1α-K197R. Experiments were performed in triplicate and data are presented as means ± SEM. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001 using two- tailed Student’s t tests.

Article Snippet: Cells transfected with HNF1α reporter plasmid pGL3-HNF4a-P2 and the control pRL-SV40 vector (E2261, Promega, USA) together with the plasmid overexpressing TRIM8 or the control plasmid were plated in 96-well plates.

Techniques: Ubiquitin Proteomics, Binding Assay, Pull Down Assay, Transfection, Expressing, Western Blot, Negative Control, Control, Luciferase, Activity Assay, Infection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Tripartite motif 8 promotes the progression of hepatocellular carcinoma via mediating ubiquitination of HNF1α

doi: 10.1038/s41419-024-06819-y

Figure Lengend Snippet: A CCK-8 assays of MHCC-L cells infected with control lentivirus or lenti-TRIM8 together with lentivirus expressing HNF1α or HNF1α-K197R. B Statistical graphs of clone formation, migration and invasion of MHCC-L cells in the indicated groups. C Images of xenograft tumors from mice inoculated with MHCC-L cells infected with control lentivirus or lenti-TRIM8, and were injected intratumorally with adenovirus expressing GFP, HNF1α or HNF1α-K197R. D Tumor volume was measured at the specified time points following subcutaneous implantation and intratumor injection. E Tumor weight was assessed when mice were sacrificed. F Representative WB analysis of HNF1α and TRIM8 protein levels in the xenograft tumors. G Semi-quantification analysis of HNF1α protein levels of xenograft tumors in the indicated groups. H The expression of HNF1α, TRIM8 and Ki67 proteins in the xenograft tumors was determined by immunohistochemistry. Scale bar = 100 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001 using non-parametric Mann–Whitney test.

Article Snippet: Cells transfected with HNF1α reporter plasmid pGL3-HNF4a-P2 and the control pRL-SV40 vector (E2261, Promega, USA) together with the plasmid overexpressing TRIM8 or the control plasmid were plated in 96-well plates.

Techniques: CCK-8 Assay, Infection, Control, Expressing, Migration, Injection, Immunohistochemistry, MANN-WHITNEY