transfections hep3b cells  (ATCC)

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

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: p62 expression is increased at protein and mRNA level upon exposure to mitochondrial and ER stress stimuli. Cells were treated for 24 h (A, C, D, E and F) or 4, 8, 24 and 48 h (B) with increasing concentrations of efavirenz (EFV), vehicle (MeOH or DMSO), thapsigargin (Tg) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM. Data (mean ± SEM) are expressed as relative protein or mRNA content in relation to that of untreated cells (control, considered 100%) after normalization with expression of the housekeeping protein (β‐actin or GAPDH) or gene (ACTB). (A) Immunoblot analysis of total cell extracts showing a representative Western blot image and summary of densitometry data of p62 expression (n = 9 except for EFV50 and rotenone n = 8, CCCP, n = 7, thapsigargin, n = 6). (B) Time‐course immunoblot analysis of total cell extracts showing representative Western blot image and densitometry data expressing quantification of p62 protein levels at the indicated treatment times (n = 7). p62 expression in untreated cells (at all time points) was considered 100%. (C) Immunoblot analysis of total cell extracts showing representative Western blot image of p62 and summary of densitometry data in ρ+ and ρ° cells (n = 6). p62 expression in untreated wild‐type (ρ+) cells was considered 100% (left panel) while p62 expression in untreated (ρ+ and ρ°; shown as Rho) cells was considered 1 (right panel). (D) Relative mRNA expression levels of SQSTM1 in Hep3B cells (n = 8, except for DMSO, thapsigargin, rotenone and CCCP n = 5) and (E) human hepatocytes (n = 2) were analysed by quantitative RT‐PCR. Data were normalized versus the housekeeping gene β‐Actin (ACTB). (F) Immunoblot analysis of total cell extracts showing representative Western blot image of p62 and summary of densitometry data in cells co‐treated with efavirenz and actinomycin D (2 μg·mL−1) (an inhibitor of gene transcription) (n = 7 except for EFV10 and EFV25, n = 5). Data (mean ± SEM) were calculated as percentage of control (untreated cells). *P < 0.05 for efavirenz; #P < 0.05 for thapsigargin, rotenone, CCCP; significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. +P < 0.05, significantly different as indicated; Student's t‐test.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: Differential sub‐cellular expression of p62. Representative Western blot image (A) of p62 and summary of densitometry data (B, C) in mitochondria‐enriched and cytosolic extracts obtained from Hep3B cells treated with increasing concentrations of efavirenz (EFV), vehicle (MeOH or DMSO), thapsigargin (Tg) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM for 24 h in the presence or absence of 2 μg·mL−1 actinomycin D (n = 5). Equal amounts of protein (30 μg) from mitochondrial and cytosolic protein extracts were loaded. Actin and porin expression was assessed to ensure the purity of the extracts. Data (mean ± SEM) were calculated as percentage of control (untreated cells were considered 100% in both extracts). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, rotenone or CCCP, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. +P < 0.05, significantly different as indicated; two‐way ANOVA followed by a Sidak's test. (D) Representative confocal fluorescence microscopy images (63× 3.0 digital zoom) of cells treated with Veh or EFV (10, 25 or 50 μM) and immunostained for p62 (red) and mitochondria (TOM20, green). Nuclei were stained with the fluorochrome Hoechst (blue). Arrows show points of colocalization. (E) Quantification of the fluorescence signal of p62 expressed as %.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Expressing, Western Blot, Control, Fluorescence, Microscopy, Staining

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: p62 expression is calcium‐ and CHOP‐dependent. Hep3B cells were treated for 24 h with increasing concentrations of efavirenz (EFV), vehicle (MeOH), thapsigargin (Tg) 2 μM or rotenone (Rot) 25 μM in the presence or absence of the inhibitor of autophagy, bafilomycin A1 20 nM. (A) Relative mRNA expression levels of SQSTM1, in presence or absence of the Ca2+ chelator BAPTA‐AM (10 μM), was analysed by quantitative RT‐PCR. Data were normalized versus the housekeeping gene β‐Actin (ACTB) and SQSTM1 expression in untreated cell (without BAPTA‐AM) was considered 100% (mean ± SEM, n = 5). (B) Representative WB image of p62, LC3 and CHOP, and summary of densitometry data in cells transfected with siControl or siCHOP. Data (mean ± SEM, n = 5) were calculated as percentage of untreated siControl cells (considered 100%). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, rotenone or CCCP, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. +P < 0.05, significantly different as indicated; Student's t‐test. (C) CHOP is associated with SQSTM1 promoter in presence of efavirenz, but not with Nrf2 or NF‐κB. A representative image of PCR bands after chromatin immunoprecipitation (ChIP) assay performed in Hep3B cells treated with efavirenz (25 μM), where chromatin was immunoprecititated with anti‐Nrf2, anti‐NF‐κB or anti‐CHOP antibody and PCR assay was carried out to amplify the immunoprecipitated chromatin using primers flanking the binding site of Nrf2, NF‐κB or CHOP on the SQSTM1 promoter (left panel); representative image of ChIP assay showing SQSTM1 promoter occupancy by CHOP in Hep3B cells treated with vehicle and efavirenz (10 and 25 μM) (right panel). A non‐related antibody anti‐IgG and input control were employed as negative and positive controls respectively.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Control

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: p62 up‐regulation occurs independently of autophagy. Hep3B cells were treated with vehicle (MeOH), EFV (10 or 25 μM), Tg 2 μM or the inhibitor of autophagy 3‐methyladenine (3MA) 2.5 mM for 24 h. (A) Representative Western blot image of LC3 and summary of densitometry data after normalization with the expression of β‐Actin in cells transiently transfected with siControl or siSQSTM1 where the expression of LC3‐II in untreated siControl cells was considered 100%. The absence of p62 confirmed the efficacy of SQSTM1 silencing. (B) Relative mRNA levels of SQSTM1, in absence or presence of 2.5 mM 3MA, were analysed by quantitative RT‐PCR and normalized versus the housekeeping gene β‐Actin (ACTB). (C) Relative mRNA levels of SQSTM1, in siControl and siATG5 cells, were analysed by quantitative RT‐PCR and normalized versus the housekeeping gene β‐Actin (ACTB). Data (mean ± SEM, n = 5) were calculated as percentage of control (untreated cells). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. n.s., no significant effects of siRNA or 3MA; Student's t‐test. (D). The expression of ATG5 in order to verify its silencing was studied by Western blotting (representative image) and the expression of GAPDH was employed as a reference.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Control

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: SQSTM1 silencing enhanced the mitochondrial dysfunction and inflammatory response induced by efavirenz but not the UPR. Hep3B cells transiently transfected with siControl or SQSTM1 were treated for 24 h with increasing concentrations of efavirenz (EFV), vehicle, thapsigargin (Tg) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM, in the absence or presence of the autophagic inhibitor 3MA. (A) Representative Western blot image of p62, CHOP and GRP78 and summary of densitometry data (n = 5). The absence of p62 confirmed the efficacy of SQSTM1 silencing. (B) Quantitative analysis of ΔΨm (tetramethylrhodamine methyl ester fluorescence) and mitochondrial superoxide production (MitoSOX fluorescence) by live cell fluorescence microscopy coupled with static cytometry (n = 7). (C) Quantitative analysis of the cell number (Hoechst fluorescence) and the mitochondrial superoxide production (MitoSOX fluorescence) by live cell fluorescence microscopy coupled with static cytometry. (D) Relative mRNA expression levels of inflammation‐related genes were analysed by quantitative RT‐PCR (n = 5). Data (mean ± SEM) were calculated as percentage of control (untreated SiControl cells). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, rotenone or CCCP, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. +P < 0.05, significantly different as indicated; Student's t‐test.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Transfection, Western Blot, Fluorescence, Microscopy, Cytometry, Expressing, Quantitative RT-PCR, Control

Journal: British Journal of Pharmacology

Article Title: Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug‐induced toxicity in vitro

doi: 10.1111/bph.14093

Figure Lengend Snippet: p62 overexpression is protective regarding cellular viability. Hep3B cells transiently transfected with Control plasmid or SQSTM1‐cherry were treated for 24 h with increasing concentrations of EFV, vehicle or thapsigargin (Tg) 2 μM. The efficacy of SQSTM1 overexpression was confirmed by WB (A). Quantitative analysis of the cell number (Hoechst fluorescence) by live cell fluorescence microscopy coupled with static cytometry (B). Data (mean ± SEM) were calculated as percentage of control (untreated Control cells). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. +P < 0.05, significantly different as indicated; Student's t‐test.

Article Snippet: Cell culture and transfection experiments Hep3B cells (human hepatoma, ATCC HB‐8064, Manassas, VA, USA) were cultured as described elsewhere (Apostolova et al., 2010 ). ρ° cells were obtained by treating wild‐type cells (8 weeks) with the mutagen, ethidium bromide (100 ng·mL −1 ), to deplete mitochondrial DNA (mtDNA) in medium supplemented with 50 μg·mL −1 uridine.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Fluorescence, Microscopy, Cytometry

Journal: Journal of cell science

Article Title: Promoter occupancy of MLL1 histone methyltransferase seems to specify the proliferative and apoptotic functions of E2F1 in a tumour microenvironment.

doi: 10.1242/jcs.126235

Figure Lengend Snippet: Fig. 4. Proliferative and pro-apoptotic activities of E2F1 in the presence of HBx. (A) ChIP-qPCR of lamin B2 origin in the lysates of HepG2 and HepG2.2.15 cells, untransfected or transfected with E2F1 DN construct to measure the occupancy of replication-associated proteins (ORC1, CDC6, MCM2, MCM4, MCM7 and Cdc45) over that of IgG control. Data are means 6 s.d. of three independent experiments; *P,0.05 and #P,0.01. (B) Western blot analysis of the DNA damage biomarker c-H2AX in the lysates of HepG2 or HepG2.2.15 cells either untransfected or transfected with E2F1 DN construct. (C) Western blot analysis of pro-apoptotic markers in the lysates extracted from scrambled (scr)-siRNA-transfected HepG2, HepG2.2.15 cells and E2F1-specific siRNA- transfected HepG2.2.15 cells. (D,E) Western blot analysis of the indicated pro-apoptotic markers in lysates of HepG2 and Hep3B cells (D) and liver tissue (E) of 3- and 6-month-old C57B6 control (C) and X15-myc transgenic (T) mice. Actin was used as loading control in these experiments.

Article Snippet: Cell culture and transfection Human hepatoma cell line Hep3B (ATCC HB-8064), hepatoblastoma cell line HepG2 (ATCC HB-8065) and its derivative HepG2.2.15 were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum.

Techniques: ChIP-qPCR, Transfection, Construct, Control, Western Blot, Biomarker Discovery, Transgenic Assay