Journal: Free radical biology & medicine

Article Title: Plumbagin is a novel GPX4 protein degrader that induces apoptosis in hepatocellular carcinoma cells.

doi: 10.1016/j.freeradbiomed.2023.03.263

Figure Lengend Snippet: Fig. 2. Plumbagin promotes GPX4 protein degradation by ubiquitin proteasome pathway in HCC. (A) Cell viability of HepG2 and Hep3B cells treated with plumbagin (PLBG, 10 μM) in the presence or absence of tetrathiomolybdate (TM, 50 μM) or deferoxamine (DFO, 20 μM) for 24 h. Mean ± SD (n = 3). ns, no significance. (B) Representative phase-contrast and PI staining images of HepG2 cells treated with PLBG (10 μM) in the presence or absence of TM (50 μM) or DFO (20 μM) for 12 h. Scale bar: 100 μm. Quantification of apoptotic cells were shown. Mean ± SD (n = 3). ns, no significance. (C,D) HepG2 and Hep3B cells were treated with indicated various concentration PLBG for 12 h or PLBG (10 μM) for indicated time periods. The protein levels of GPX4, catalase (CAT), superoxide dismutase 1 (SOD1), and thioredoxin (TXN) were measured by western blotting. GAPDH was used as a loading control. The bands were quantified and normalized to GAPDH. Numbers shown below the bands represent quantification of corresponding proteins. (E) HepG2 cells were treated with PLBG with or without bortezomib (BTZ, 50 nM) for 24 h. The protein expression of GPX4 was assessed by western blotting.

Article Snippet: Human hepatocellular carcinoma (HCC) cells HepG2 and Hep3B were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Staining, Concentration Assay, Western Blot, Control, Expressing

Journal: Free radical biology & medicine

Article Title: Plumbagin is a novel GPX4 protein degrader that induces apoptosis in hepatocellular carcinoma cells.

doi: 10.1016/j.freeradbiomed.2023.03.263

Figure Lengend Snippet: Fig. 4. Plumbagin acts as a DUB inhibitor and in creases GPX4 ubiquitination. (A) Deubiquitinating enzymes (DUBs) activity of HepG2 and Hep3B cells treated with various concentration plumbagin (PLBG) or a DUB inhibitor N-Ethylmaleimide (NEM, 2 mM) for 3 h. Mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) HepG2 and Hep3B cells were treated with PLBG (10 μM) for indicated time periods or apoptosis inducer staurosporine (STA, 1 μM) for 24 h. The protein levels of PARP, cleaved caspase 3 (CC3) and ubiquitinated proteins (Ub) were assessed by western blotting. GAPDH was used as a loading control. (C) HepG2 and Hep3B cells were treated with PLBG for 24 h and MG132 (10 μM) for the last 6 h. The extracts were immunoprecipitated with anti-GPX4 antibodies and immunoblotted with anti-ubiquitin (Ub) antibodies.

Article Snippet: Human hepatocellular carcinoma (HCC) cells HepG2 and Hep3B were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Activity Assay, Concentration Assay, Western Blot, Control, Immunoprecipitation

Journal: Free radical biology & medicine

Article Title: Plumbagin is a novel GPX4 protein degrader that induces apoptosis in hepatocellular carcinoma cells.

doi: 10.1016/j.freeradbiomed.2023.03.263

Figure Lengend Snippet: Fig. 5. Plumbagin may decrease GPX4 protein by inhibiting USP31. (A) The extracts from HepG2 cells were immunoprecipitated with anti-GPX4 or IgG an tibodies and immunoblotted with anti-GPX4 anti bodies or various anti-DUBs antibodies. (B) The extracts from Hep3B cells were immunoprecipitated with anti-GPX4 or IgG antibodies and immunoblotted with anti-GPX4, anit-USP31, anti-USP14, or anti- UCHL5 antibodies. (C) HepG2 cells transfected USP31 or vector plasmids were treated with or without plumbagin (PLBG, 10 μM) for 24 h. The expression of USP31 and GPX4 was assessed by western blotting. Actin was used as a loading control. (D) HepG2 cells transfected USP31 or vector plasmids were treated with or without PLBG (10 μM) for 24 h. Representative phase-contrast and PI staining images, and quantification of apoptotic cells were shown. Scale bar: 100 μm. Mean ± SD (n = 3). ns, no sig nificance, *p < 0.01, ***p < 0.001.

Article Snippet: Human hepatocellular carcinoma (HCC) cells HepG2 and Hep3B were obtained from ATCC.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Staining

Journal: Nucleic acids research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: Figure 2. A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Tw enty -f our hours af ter knoc kdo wn, cells w ere transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assa y s after mouse A-Myb o v ere xpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assa y s with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 / M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANO V A (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cell culture and cell count analysis Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Lonza).

Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 expression is elevated in HCC cells (A) UCSC database presented PRR34-AS1 expression profile in multiple human normal tissues, including liver tissues. (B) NONCODE database indicated the profile of PRR34-AS1 expression in different normal tissue samples. (C) GEPIA2 database exhibited PRR34-AS1 expression pattern in 369 cases of LIHC tissues and 160 cases of normal tissues. (D) Quantitative real-time RT-PCR analyzed PRR34-AS1 expression in HCC cell lines (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and human normal hepatocyte cell line (THLE-3). (E and F) Subcellular fractionation and FISH experiments determined PRR34-AS1 location in HCC cells. (G) Knockdown efficiencies of sh-PRR34-AS1#1&2&3 in MHCC97-H and HCCLM3 cells, as well as overexpression efficiency of pcDNA3.1/PRR34-AS1 in Hep 3B cells, were evaluated via quantitative real-time RT-PCR. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Fractionation, Knockdown, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 facilitates HCC cell proliferation in vitro and tumor growth in vivo (A) CCK-8 assays tested the changes in cell proliferation ability induced by PRR34-AS1 silencing in MHCC97-H and HCCLM3 cells and induced by PRR34-AS1 upregulation in Hep 3B cells. (B) Colony formation assays measured the number of colonies formed under PRR34-AS1 silencing in MHCC97-H, HCCLM3 cells, or under PRR34-AS1 upregulation in Hep 3B cells. (C and D) TUNEL assays and flow cytometry analysis assessed the apoptosis rate when PRR34-AS1 was downregulated in HCCLM3 cells (E) Representative pictures of xenografts derived from HCCLM3 cells transfected with sh-NC or sh-PRR34-AS1#1. (F) The grow curves of tumors from two different groups. (G and H) The final tumor volume and weight were measured. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, In Vivo, CCK-8 Assay, TUNEL Assay, Flow Cytometry, Derivative Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 contributes to cell migration, invasion, and EMT process in HCC cells and activates the Wnt/β-catenin pathway (A) Wound healing assays detected the migration ability of indicated HCC cells when PRR34-AS1 was knocked down or upregulated. (B and C) Transwell experiments analyzed the migration and invasion properties in PRR34-AS1 silenced MHCC97-H and HCCLM3 cells, as well as in PRR34-AS1 upregulated Hep 3B cells. (D) Western blot assay examined the protein levels of E-cadherin, N-cadherin, and Vimentin in HCC cells under PRR34-AS1 depletion or overexpression. (E) H&E staining evaluated the metastatic ability of HCCLM3 cells after PRR34-AS1 depletion. (F) TOP Flash/FOP Flash reporter assays assessed the activity of Wnt/β-catenin in HCC cells with PRR34-AS1 depletion or overexpression. (G–I) Western blot evaluated the levels of indicated proteins in HCC cells after PRR34-AS1 depletion or overexpression. ∗p < 0.05.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Western Blot, Over Expression, Staining, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 sponges miR-296-5p in HCC cells (A) LncBase database was used to predict underlying miRNAs that could combine with PRR34-AS1. (B) RNA pull-down assays assessed the abundance of candidate miRNAs in biotinylated PRR34-AS1 group in Hep 3B cells. (C) ENCORI database predicted the underlying binding sites between PRR34-AS1 and miR-296-5p. (D) Quantitative real-time RT-PCR analyzed miR-296-5p expression pattern in HCC cells (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and human normal hepatocyte cells (THLE-3). (E) RIP assays assessed the abundance of PRR34-AS1 and miR-296-5p in AGO2 complex. (F) Luciferase reporter assays detected the luciferase activity of PRR34-AS1-WT or PRR34-AS1-Mut under miR-296-5p upregulation in HCC cells. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 aggravates HCC cell proliferation, migration, and EMT through targeting miR-296-5p (A) Quantitative real-time RT-PCR detected the expression of miR-296-5p in HCCLM3 cells under indicated transfections. (B and C) CCK-8 and colony formation assays examined the proliferation ability of indicated HCCLM3 cells. (D and E) TUNEL and flow cytometry experiments analyzed the apoptosis rate of indicated HCCLM3 cells. (F–H) Wound healing and Transwell assays detected the migratory and invasive capacities of indicated HCCLM3 cells. (I) Western blot examined the protein levels of E-cadherin, N-cadherin, and Vimentin in HCCLM3 cells under different conditions. ∗p < 0.05.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, TUNEL Assay, Flow Cytometry, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: E2F2 and SOX12 are identified as the targets of miR-296-5p in HCC cells (A) The mirDIP, microT, PITA, and miRmap databases were used to screen underlying mRNAs that potentially combined with miR-296-5p. (B) Quantitative real-time RT-PCR analyzed the expression of candidate targets (ZNF76, HMGA1, FAM53B, FGFR3, E2F2, HIPK1, SOX12, CDK16, BMF, and SLC16A3) in PRR34-AS1 silenced HCCLM3 cells. (C) ENCORI predicted the binding sites between E2F2/SOX12 and miR-296-5p. (D) Quantitative real-time RT-PCR detected E2F2 and SOX12 mRNA levels in HCC cells (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and THLE-3 cells. (E) Western blot assay analyzed the protein levels of E2F2 and SOX12 in PRR34-AS1 silenced or overexpressed HCC cells. (F) RIP assays assessed the enrichments of PRR34-AS1, miR-296-5p, and E2F2/SOX12 in anti-AGO2 complex. (G) Luciferase reporter experiments assessed the luciferase activity of E2F2-3′ UTR-WT/Mut and SOX12-WT/Mut in HEK293T cells with different transfections. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Western Blot, Luciferase, Activity Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: E2F2 activates PRR34-AS1 transcription in HCC cells (A) Western blot analyzed the knockdown or overexpression efficiencies of E2F2 in HCC cells. (B and C) The levels of E2F2 and PRR34-AS1 in indicated HCC cells were examined via quantitative real-time RT-PCR. (D) ChIP experiments measured the connection between PRR34-AS1 promoter and E2F2 in HCC cells. (E) The binding motif of E2F2 were predicted by JASPAR. (F) Luciferase reporter experiments assessed the luciferase activities of PRR34-AS1-Pro-WT and PRR34-AS1-Pro-Mut in HCC cells after E2F2 expression was increased or silenced. ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Western Blot, Knockdown, Over Expression, Quantitative RT-PCR, Binding Assay, Luciferase, Expressing

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Western blot analysis of normoxic (N) or hypoxic (H) Hep3B cells. ( a ) Representative result of n =3 independent experiments. Protein masses are given in kDa. ( b ) Densitometry of ARNT protein levels. The ratio of ARNT/Actin was calculated and normalised to normoxic control cells. Values are presented as mean±S.E.M. of n =3 independent experiments. Statistical analysis was done using the one-sample t -test

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Western Blot, Control

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: qRT-PCR analysis of ARNT mRNA expression in Hep3B cells cultured in normoxia or hypoxia for 2, 5 and 8h, respectively. ARNT mRNA was measured using the ΔΔC t method relative to B2M expression (endogenous control). ARNT mRNA expression was normalised to appropriate normoxic controls. Each graph represents the results of n =3 independent experiments. Values are given as mean±S.E.M. Statistical analysis was conducted using the one-sample t -test. Appropriate P -values are indicated

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Control

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Knockdown and overexpression studies in Hep3B cells. ( a ) Western blot analysis of siRNA-transfected cells exposed to normoxia or hypoxia for 8 h. The Arrow indicates the specific signal for HIF-2 α . Protein masses are given in kDa. ctrl.: control siRNA-transfected cells. Representative result of n =3 independent experiments. ( b ) Densitometric analysis of ARNT protein expression in knockdown cells (corresponding to a ). The ARNT/ α -Tubulin ratio was calculated and normalised to normoxic control cells. Values are presented as mean±S.E.M. of n =3 independent experiments. ( c ) Western blot analysis of transiently transfected cells using increasing amounts of the HIF-1 α expression vector (pHIF-1 α ) in conjunction with normoxic (N) and hypoxic (H) conditions. Protein masses are given in kDa. ctrl.: control transfected cells (pcDNA3, 2 μ g/well). Representative result of n =3 independent experiments. ( d ) Densitometric analysis of ARNT protein expression in HIF-1 α- overexpressing cells (corresponding to c ). The ARNT signal was set in ratio to the appropriate loading control and normalised to normoxic control cells. Values are presented as mean±S.E.M. of n =3 independent experiments

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Knockdown, Over Expression, Western Blot, Transfection, Control, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Cycloheximide (CHX) chase experiments in transfected Hep3B cells in normoxia. ( a ) Western blot analysis of HIF-1 α and ARNT degradation after CHX treatment (100 μ M) in control (ctrl.) and pHIF-1 α- transfected cells. Protein masses are given in kDa. Representative result of n =3 independent experiments. ( b ) Quantitation of ARNT degradation. Values represent mean±S.E.M. of n =3 (except ctrl. 1 h: n =2) independent experiments; lin. reg.: linear regression. ( c ) Quantitation of HIF-1 α degradation in pHIF-1 α -transfected cells. Values represent mean±S.E.M. of n =3 independent experiments

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Transfection, Western Blot, Control, Quantitation Assay

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Effects on transcription. ( a ) Treatment of Hep3B cells with Actinomycin D (Act D). Cells were incubated with increasing concentrations of Act D in normoxia (N) and hypoxia (H, 3% O 2 ) for 8 h as indicated. DMSO at the highest concentration (0.1 v/v %) was used as vehicle control. HIF-1 α and ARNT protein levels were determined by western blot analysis. Representative result of n =3 independent experiments. ( b ) Densitometry of ARNT protein levels (corresponding to a). The ratio of ARNT/ α -Tubulin was calculated and normalised to normoxic vehicle-treated control cells. Values are given as mean±S.E.M. of n =3 independent experiments. ( c ) Knockdown of HIF-1 α in hypoxic Hep3B cells. Cells were transfected either with control (ctrl.) or HIF-1 α siRNA (siHIF-1 α ) and exposed to 3% O 2 for 5 h. HIF-1 α and ARNT mRNA expression were measured by qRT-PCR as described in materials and methods. Normalised expression levels are shown. Values are presented as mean±S.E.M. of n =3 independent experiments. Statistical comparison was performed using the one-sample t -test. Appropriate P- values are indicated

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Incubation, Concentration Assay, Control, Western Blot, Knockdown, Transfection, Expressing, Quantitative RT-PCR, Comparison

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Overexpression of ARNT in Hep3B cells. ctrl.: control (pcDNA3, 2.5 μ g/well) transfected cells. Representative result of n =3 independent experiments

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Over Expression, Control, Transfection

Journal: Cell Death & Disease

Article Title: A HIF-1 α -driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT

doi: 10.1038/cddis.2016.187

Figure Lengend Snippet: Re-oxygenation of control and ARNT-overexpressing Hep3B cells. Transiently transfected control (ctrl.) or ARNT-overexpressing (pARNT) cells were incubated in hypoxia (3% O 2 ) for 8 h and subsequently exposed to normoxia for indicated time points followed by western blot analysis. Representative result of n =3 independent experiments

Article Snippet: Human hepatocellular carcinoma Hep3B cells (ATCC) were maintained in RPMI 1640 medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin.

Techniques: Control, Transfection, Incubation, Western Blot

Journal: Cancer Communications

Article Title: Galectin 3‐binding protein (LGALS3BP) depletion attenuates hepatic fibrosis by reducing transforming growth factor‐β1 (TGF‐β1) availability and inhibits hepatocarcinogenesis

doi: 10.1002/cac2.12600

Figure Lengend Snippet: Regulation of TGFB1 expression by JunB with rLGALS3BP treatment. (A) Identification of enriched transcription factor binding motifs with unique chromatin‐accessible peaks in the vehicle‐ and rLGALS3BP‐treated cells from ATAC‐seq. (B) JUN/AP‐1 luciferase assays using indicated HCC cell lines with rLGALS3BP treatment for 6h. Data represented as mean ± SD, n = 3. Data represented as mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001 (Student's t ‐test to non‐treated controls). (C) Chromatin accessibility of the TGFB1 locus. The locations of the three JunB‐binding motifs are indicated, and arrows denote the amplicons of the JunB‐binding sites of TGFB1 . (D) JunB ChIP‐qPCR for TGFB1 JunB‐RE locus using Hepa‐1c1c7 cells upon rLGALS3BP treatment for 6h. The UNTR4 region served as the negative control. Data represented as mean ± SD, n = 3. *** P < 0.001 (Student's t ‐test). (E) qRT‐PCR results (upper) and western blot analysis (lower) of Hepa‐1c1c7 cells. Cells were transfected with JUNB‐specific siRNA or a negative control siRNA for 24h then serum‐starved before treated with rLGALS3BP for 6h. Data represented as mean ± SD, n = 3. *** P < 0.001 (Student's t ‐test). (F) qRT‐PCR to measure JUNB expression of the control and LGALS3BP ‐KI primary hepatocytes. Data represented as mean ± SD, n = 3. *** P < 0.001 (Student's t ‐test). (G) The correlation between LGALS3BP and JUNB in HCC from TCGA dataset ( n = 366). (H) The correlation between LGALS3BP and JUNB in tumoral tissues of HCC patients at CNUHH ( n = 83). (I) The correlation between LGALS3BP and JUNB in peri‐tumoral normal tissues of HCC patients at CNUHH ( n = 83). * P < 0.05, ** P < 0.01, and *** P < 0.001 (Two‐way ANOVA). Abbreviations: ATAC‐seq, the assay for transposase‐accessible chromatin with sequencing; ATF4, activating transcription factor 4; Cont, control; FOSL2, FOS like 2; FOXA1, forkhead box protein A1; FOXO3, forkhead box protein O3; JUNB‐RE, JunB response elements; KI, LGALS3BP knockin; rLGALS3BP, recombinant LGALS3BP; TEAD4, TEA domain transcription factor 4; UNTR6 , untranscribed region 6.

Article Snippet: Mouse HCC (Hepa‐1c1c7) and human HCC cells (HepG2, Hep3B and SNU449) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) or Korean cell line bank (KCLB, Seoul, South Korea).

Techniques: Expressing, Binding Assay, Luciferase, ChIP-qPCR, Negative Control, Quantitative RT-PCR, Western Blot, Transfection, Control, Sequencing, Knock-In, Recombinant

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 1. TANGO1 expression in HCC samples A, B, C. TCGA database presented the increase of TANGO1 in HCC samples. D. Statistical results of TANGO1 expression and HIC score. E. Western blot detection TANGO1 expression in cell lines. F. Kaplan-Meier showing TANGO1 expression in TMA survival analysis. G. shRNA-TANGO1 and control lentivirus transfection HepG2 and Huh7. H. TANGO1 and vector lentivirus transfection HepG2 and Huh7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Expressing, Western Blot, shRNA, Control, Transfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 3. TANGO1 promotes HCC cell progression and inhibits apoptosis A, B. Cell cycle analysis of HepG2 and Huh7. C, D. Apoptosis levels of HepG2 and Huh7. E. Expression of cell cycle-related proteins verified by Western blot. *p < 0.05, **p < 0.01.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Cell Cycle Assay, Expressing, Western Blot

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 5. TANGO1-interacting proteins and pathway enrichment A. RNA-seq of TANGO1 overexpression HepG2, volcano plot of DEGs obtained. B. GO functional enrichment to differential genes of BP, CC, MF. C. Biological pathways screened by KEGG functional analysis. D. qPCR validation of partial differential gene expression in TANGO1 overexpressing Huh7. E. TCGA database presented the increase of NRTN in HCC samples. F. Kaplan-Meier analysis of NRTN overall survival in HCC patients. *p < 0.05, **p < 0.01.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: RNA Sequencing, Over Expression, Functional Assay, Biomarker Discovery, Gene Expression

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 6. TANGO1 regulates the PI3K/AKT/mTOR pathway in HCC cells and interacts with NRTN A. Co-IP assay TANGO1/NRTN interaction in HepG2 and Huh7. B. Co-localization of TANGO1 and NRTN in HepG2 and Huh7. C, D. Western blot detect PI3K/AKT/mTOR pathway in HepG2 and Huh7. **p < 0.01, ***p < 0.001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Biochemical pharmacology

Article Title: TANGO1 interacts with NRTN to promote hepatocellular carcinoma progression by regulating the PI3K/AKT/mTOR signaling pathway.

doi: 10.1016/j.bcp.2023.115615

Figure Lengend Snippet: Fig. 7. NRTN silencing inhibits TANGO1-mediated proliferation and migration A. Western blot assay NRTN expression after siRNA transfection of HepG2 and Huh7. B. EdU assay showing silencing of NRTN inhibits HepG2 and Huh7 proliferation. C Wound healing assay showing silencing of NRTN inhibits HepG2 and Huh7 migration. D. Transwell assay showing silencing of NRTN inhibited HepG2 and Huh7 invasion. E. Western blot assay PI3K/AKT/mTOR marker expression in HepG2 and Huh7 after NRTN silencing. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: human liver (L02) and HCC (Hep3B, HepG2, Huh7, LM3) cell line (Pricella); fetal bovine serum (FBS); penicillin–streptomycin, trypsin, DMEM (Gibco); Transwell chambers (Corning), N-cadherin, Vimentin, E-cadherin, p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, β-tubulin, GAPDH, cyclin D1, Cleaved caspase-3, PCNA, Ki67 antibodies (Proteintech); TANGO1(Abcam, Santa Cruz Biotechnology), p-PI3K (Abcam); NRTN (Bioss, Santa Cruz Biotechnology); mouse-derived secondary antibody, rabbit-derived secondary antibody (Abcam); TRIzol kit (Nitrogen); immunoprecipitation kit, cDNA synthesis kit (Thermo Scientific); BCA working solution, RIPA lysis solution, PMSF, prestained Marker (Solarbio); CCK-8 kit ECL working solution, Matrigel gel, SDSPAGE gel-making kit, cell cycle assay kit, Annexin VFITC/PI cell regulation assay kit (Beyotime); NRTN small interfering RNA and negative control (Genechem); TANGO1 overexpression lentivirus and negative control, TANGO1 knockdown lentivirus and negative control (Huabio); NU/NU nude mice (Vital River) were used in this study.

Techniques: Migration, Western Blot, Expressing, Transfection, EdU Assay, Wound Healing Assay, Transwell Assay, Marker

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 1. High glucose increases O-GlcNAcylation of DNMT1 in cell lines and primary cells. (A) Hep3B cells were treated with glucose (5 mM or 25 mM) with or without Thiamet-G (TMG). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). (B) Lysates of Hep3B treated with glucose were immunoprecipitated with DNMT1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc (n = 3). (C) Peripheral blood mononuclear cells (PBMCs) were isolated from three individual donor blood samples and treated with increasing concentration of glucose for 24 hr. Collected cell lysates from PBMCs were immunoprecipitated with antibody targeting DNMT1 and immunoblotted for O-GlcNAc. Representative blot from one donor (n = 3). (D) Immunoblots for O-GlcNAc and GAPDH from liver samples of C57BL/6J mice given a high- fat/high-sucrose diet (HF/HS) or normal diet (chow) for 4 mo, and immunoprecipitated with Dnmt1. Lysates of mouse liver were immunoprecipitated with Dnmt1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc. *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A-D); ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: DOI: https://doi.org/10.7554/eLife.85595 14 of 23 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Commercial assay or kit EpiQuik DNMT Activity/Inhibition ELISA Easy Kit EpiGentek Cat# P- 3139 - Commercial assay or kit Global DNA methylation LINE- 1 Active Motif Cat# 55017 - Commercial assay or kit EpiQuik 8- OHdG DNA Damage Quantification Direct kit EpiGentek Cat# P- 6003 - Chemical compound, drug OSMI- 4 Selleck Chem Cat# S8910 - Chemical compound, drug Thiamet- G (TMG) Cayman Chemical Cat# 13237 - Software, algorithm bedGraphToBigWig Kent et al., 2010 - - Software, algorithm Clustal Omega Sievers et al., 2011 - Version 1.2.4 Software, algorithm GraphPad Prism 9 GraphPad - Version: 9.3.1 Software, algorithm Minimap2 Li and Birol, 2018 RRID:SCR_018550 Version: 2.17 Software, algorithm Nanopolish Loman et al., 2015 RRID:SCR_016157 Version: 0.11.1 Software, algorithm Python Python Core Team Version: 3.8.2 Software, algorithm R R Core Team - Version: 3.4.3 Software, algorithm Samtools Lister et al., 2009 RRID:SCR_002105 Version: 1.10 Other UniProt The UniProt Consortium - Database of protein information (https://www. uniprot.org/) Other DAPI stain Invitrogen D1306 1 μg/ml; marker for nuclear DNA Continued Cell culture and plasmid DNA transfection Human hepatocellular carcinoma cell lines Hep3B (HB- 8064) and HepG2 (HB- 8065) were purchased from ATCC (Manassas, VA).

Techniques: Western Blot, Immunoprecipitation, Isolation, Concentration Assay

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 2. Identification of O-GlcNAcylated sites within DNMT1 by LC-MS/MS. (A) Schematic drawing of the DNMT1 O-GlcNAc-modified region enriched from Hep3B cells based on mass spectrometry (MS) data and tandem MS (MS/MS) peaks. FTMS+ p NSI full MS (400.0000–1600.0000). DQDYARFESPPKTQPTEDNKF (S9 HexNAc) – S878. (B) Schematic diagram of identified novel O-GlcNAcylated and phosphorylated sites within DNMT1 as determined via LC-MS/MS. DMAP, DNA methyltransferase associated protein-binding domain; PCNA, proliferating cell nuclear antigen-binding domain; NLS, nuclear localization sequences; RFTS, replication foci targeting sequence domain; BAH, bromo-adjacent homology domain. (C) Sequence conservation of S878 in vertebrates. (D) Each immunoprecipitated Myc-DNMT1 wild type and substituted mutants was immunoblotted with an O- GlcNAc antibody (n = 3). **p<0.0005; ***p<0.0001 by Student’s t-test (D); N.D., not detected, ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: DOI: https://doi.org/10.7554/eLife.85595 14 of 23 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Commercial assay or kit EpiQuik DNMT Activity/Inhibition ELISA Easy Kit EpiGentek Cat# P- 3139 - Commercial assay or kit Global DNA methylation LINE- 1 Active Motif Cat# 55017 - Commercial assay or kit EpiQuik 8- OHdG DNA Damage Quantification Direct kit EpiGentek Cat# P- 6003 - Chemical compound, drug OSMI- 4 Selleck Chem Cat# S8910 - Chemical compound, drug Thiamet- G (TMG) Cayman Chemical Cat# 13237 - Software, algorithm bedGraphToBigWig Kent et al., 2010 - - Software, algorithm Clustal Omega Sievers et al., 2011 - Version 1.2.4 Software, algorithm GraphPad Prism 9 GraphPad - Version: 9.3.1 Software, algorithm Minimap2 Li and Birol, 2018 RRID:SCR_018550 Version: 2.17 Software, algorithm Nanopolish Loman et al., 2015 RRID:SCR_016157 Version: 0.11.1 Software, algorithm Python Python Core Team Version: 3.8.2 Software, algorithm R R Core Team - Version: 3.4.3 Software, algorithm Samtools Lister et al., 2009 RRID:SCR_002105 Version: 1.10 Other UniProt The UniProt Consortium - Database of protein information (https://www. uniprot.org/) Other DAPI stain Invitrogen D1306 1 μg/ml; marker for nuclear DNA Continued Cell culture and plasmid DNA transfection Human hepatocellular carcinoma cell lines Hep3B (HB- 8064) and HepG2 (HB- 8065) were purchased from ATCC (Manassas, VA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Modification, Mass Spectrometry, Tandem Mass Spectroscopy, Protein Binding, Binding Assay, Sequencing, Immunoprecipitation