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ATCC cell lines hep3b atcc hb 8064 hepg2 atcc hb 8065 oligonucleotides primers
Cell Lines Hep3b Atcc Hb 8064 Hepg2 Atcc Hb 8065 Oligonucleotides Primers, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 29980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Mutagenesis:

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity
Article Snippet: hepatocarcinoma Hep3B cell lines were respectively provided by the EGID (European Genomic Institute for Diabetes) and ONCOLille institutes in Lille. .. HepG2 and Hep3B cell lines are ATCC certified. .. Both cell lines, tested and free from mycoplasma , were cultured in Minimal Essential Medium (MEM, Biowest) supplemented with 2 mM L-glutamine, 1 mM s

Expressing:

Membrane:

Transfection:

Plasmid Preparation:

Western Blot:

Cell Fractionation:

Fractionation:

Clear Native PAGE:

Activity Assay:

Incubation:

Marker:

Software:

Confocal Microscopy:

Staining:

MTS Assay:

Cell Counting:

Flow Cytometry:

Modification:

Image Search Results

$Mutation of the T980 O -GlcNAcylation site reduces FASN expression, membrane association, homodimerization, and stability . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Cell lysates were analyzed by Western blot (n = 8) by using an anti-Flag antibody ( A ). Molecular mass markers are indicated on the left (kDa). Optical densities were measured and normalized with β-actin expression. B , cytosolic and membrane-associated 3xFlag-FASN contents were prepared by cell fractionation and analyzed by Western blot (n = 3). Fractionation efficiency was assessed using anti-GAPDH and anti-E-Cadherin antibodies for cytosolic and membrane fractions, respectively. Optical densities were measured and normalized with these markers. C , cell lysates were analyzed by Native-PAGE (n = 3) according to their 3xFlag-FASN monomers (∼250 kDa) and dimers (∼500 kDa) contents. Optical densities were measured and normalized with total protein. D , CHX chase assay was performed by treating cells with 40 μg/ml CHX for the indicated time periods (0–24 h). Data are presented with means ± SD. ∗p < 0.05; ∗∗ p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.$

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: Mutation of the T980 O -GlcNAcylation site reduces FASN expression, membrane association, homodimerization, and stability . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Cell lysates were analyzed by Western blot (n = 8) by using an anti-Flag antibody ( A ). Molecular mass markers are indicated on the left (kDa). Optical densities were measured and normalized with β-actin expression. B , cytosolic and membrane-associated 3xFlag-FASN contents were prepared by cell fractionation and analyzed by Western blot (n = 3). Fractionation efficiency was assessed using anti-GAPDH and anti-E-Cadherin antibodies for cytosolic and membrane fractions, respectively. Optical densities were measured and normalized with these markers. C , cell lysates were analyzed by Native-PAGE (n = 3) according to their 3xFlag-FASN monomers (∼250 kDa) and dimers (∼500 kDa) contents. Optical densities were measured and normalized with total protein. D , CHX chase assay was performed by treating cells with 40 μg/ml CHX for the indicated time periods (0–24 h). Data are presented with means ± SD. ∗p < 0.05; ∗∗ p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Mutagenesis, Expressing, Membrane, Transfection, Plasmid Preparation, Western Blot, Cell Fractionation, Fractionation, Clear Native PAGE

FASN T980 is crucial for FASN activity . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors and incubated with the lipid droplet marker BODIPY 493/503 (n = 3) ( A ). Lipid droplets were counted using the ImageJ software ( B ). Confocal microscopy analysis indicates that FASN T980A mutation significantly impacts lipid droplets formation (bar size: 15 μm). Data are presented with means ± SD. ∗ p < 0.05.

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for FASN activity . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors and incubated with the lipid droplet marker BODIPY 493/503 (n = 3) ( A ). Lipid droplets were counted using the ImageJ software ( B ). Confocal microscopy analysis indicates that FASN T980A mutation significantly impacts lipid droplets formation (bar size: 15 μm). Data are presented with means ± SD. ∗ p < 0.05.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Incubation, Marker, Software, Confocal Microscopy, Mutagenesis

FASN O -GlcNAcylation at T980 is pivotal for Hep3B cells survival, proliferation, and cell cycle progression. Low-density Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Six days later, colonies were fixed and stained with crystal violet (n = 3) ( A ). Using MTS assay ( B ), cell survival was evaluated according to densitometry measured at λ = 490 nm (n = 6). Cell proliferation was determined by cell counting (n = 4) ( C ). Cell cycle was analyzed by flow cytometry (n = 3) ( D ). Data are presented with means ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN O -GlcNAcylation at T980 is pivotal for Hep3B cells survival, proliferation, and cell cycle progression. Low-density Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Six days later, colonies were fixed and stained with crystal violet (n = 3) ( A ). Using MTS assay ( B ), cell survival was evaluated according to densitometry measured at λ = 490 nm (n = 6). Cell proliferation was determined by cell counting (n = 4) ( C ). Cell cycle was analyzed by flow cytometry (n = 3) ( D ). Data are presented with means ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, MTS Assay, Cell Counting, Flow Cytometry

FASN T980 is crucial for various properties of Hep3B cells. The O -GlcNAcylation at T980 is crucial for FASN expression, stability, membrane residence, homodimerization, and activity, promoting Hep3B cells survival, proliferation, and cell cycle progression. This highlights the O -GlcNAcylation of FASN at T980 as a potential key modification that supports hepatic carcinogenesis.

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for various properties of Hep3B cells. The O -GlcNAcylation at T980 is crucial for FASN expression, stability, membrane residence, homodimerization, and activity, promoting Hep3B cells survival, proliferation, and cell cycle progression. This highlights the O -GlcNAcylation of FASN at T980 as a potential key modification that supports hepatic carcinogenesis.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Expressing, Membrane, Activity Assay, Modification

Functional characterization of IL-15 +/+ /TGFβR2 −/− iNK cells (A) WT iNK cells and IL-15 +/+ /TGFβR2 −/− iNK cells were plated with the same number in NK expansion medium with or without IL-2. After 3 days (D3) or 7 days (D7), cells were collected and analyzed by flow cytometry with quantitative beads. Error bars represent mean ± SD. ∗ p < 0.05 by t test; ∗∗∗ p < 0.001 by t test. (B) WT (left) and IL-15 +/+ /TGFβR2 −/− iNK cells (right) were cultured under normal conditions (Ctrl) or in the presence of TGF-β1. The normalized MFIs of the staining of NK-activating receptors, including NKG2D, DNAM-1, NKp30, CD16, and NKp46, were measured by flow cytometry analysis. The expression level of CD2 was used as a control. (C) The IL-15 +/+ /TGFβR2 −/− iNK cells are resistant to TGF-β-mediated suppression. The killing activity against Hep3B HCC tumor cells was compared between WT iNK cells and IL-15 +/+ /TGFβR2 −/− iNK cells under normal conditions (Ctrl) or in the presence of TGF-β1. Error bars represent mean ± SD. (D) IL-15 +/+ /TGFβR2 −/− iNK cells were used as effectors at an E:T ratio of 1:1 with K562 myeloid leukemia cells. The non-edited iNK cells (WT iNKs) were used as control effectors, while K562 cells alone were used as reference. Data are representative of at least two independent experiments with each tumor cell line and are shown as means ± SDs ( n = 6 technical replicates).

Journal: Cell Reports Methods

Article Title: An improved approach to generate IL-15 +/+ /TGFβR2 −/− iPSC-derived natural killer cells using TALEN

doi: 10.1016/j.crmeth.2024.100857

Figure Lengend Snippet: Functional characterization of IL-15 +/+ /TGFβR2 −/− iNK cells (A) WT iNK cells and IL-15 +/+ /TGFβR2 −/− iNK cells were plated with the same number in NK expansion medium with or without IL-2. After 3 days (D3) or 7 days (D7), cells were collected and analyzed by flow cytometry with quantitative beads. Error bars represent mean ± SD. ∗ p < 0.05 by t test; ∗∗∗ p < 0.001 by t test. (B) WT (left) and IL-15 +/+ /TGFβR2 −/− iNK cells (right) were cultured under normal conditions (Ctrl) or in the presence of TGF-β1. The normalized MFIs of the staining of NK-activating receptors, including NKG2D, DNAM-1, NKp30, CD16, and NKp46, were measured by flow cytometry analysis. The expression level of CD2 was used as a control. (C) The IL-15 +/+ /TGFβR2 −/− iNK cells are resistant to TGF-β-mediated suppression. The killing activity against Hep3B HCC tumor cells was compared between WT iNK cells and IL-15 +/+ /TGFβR2 −/− iNK cells under normal conditions (Ctrl) or in the presence of TGF-β1. Error bars represent mean ± SD. (D) IL-15 +/+ /TGFβR2 −/− iNK cells were used as effectors at an E:T ratio of 1:1 with K562 myeloid leukemia cells. The non-edited iNK cells (WT iNKs) were used as control effectors, while K562 cells alone were used as reference. Data are representative of at least two independent experiments with each tumor cell line and are shown as means ± SDs ( n = 6 technical replicates).

Article Snippet: Hep3B cell line , ATCC , HB-8064.

Techniques: Functional Assay, Flow Cytometry, Cell Culture, Staining, Expressing, Control, Activity Assay

Journal: Cell Reports Methods

Article Title: An improved approach to generate IL-15 +/+ /TGFβR2 −/− iPSC-derived natural killer cells using TALEN

doi: 10.1016/j.crmeth.2024.100857

Figure Lengend Snippet:

Article Snippet: Hep3B cell line , ATCC , HB-8064.

Techniques: Recombinant, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, DNA Purification, Mutagenesis, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software, Flow Cytometry

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.

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

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.

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.

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

Figure 2. The PPM1K-pBCKDHA axis acts as a glutamine-deprivation-induced BCAA catabolic switch (A) Schematic diagram of the BCAA catabolic pathway with major metabolites and enzymes in the process. (B) Western blot analysis of BCAT1/2, BCKDHA/B, pBCKDHA, BCKDK, and PPM1K in Hep3B cells cultured under normal, glucose-starvation, or glutamine- starvation conditions for 48 h. (C) Western blot analysis of pBCKDHA expression in Hep3B cells when PPM1K was knocked down under glutamine-deprivation conditions. (D) Detection of BCKDHA and PPM1K knockdown efﬁciency in Hep3B stable cell lines under glutamine-starvation conditions. (E and F) Hep3B cells knocking down BCKDHA or PPM1K were labeled under glutamine-starvation conditions and measured by HRMS after 48 h. Percentages of 15N labeling in NEAAs and UMP (E). Percentages of 13C labeling in TCA intermediate metabolites (F).

Journal: Cell reports

Article Title: Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

doi: 10.1016/j.celrep.2022.111691

Figure Lengend Snippet: Figure 2. The PPM1K-pBCKDHA axis acts as a glutamine-deprivation-induced BCAA catabolic switch (A) Schematic diagram of the BCAA catabolic pathway with major metabolites and enzymes in the process. (B) Western blot analysis of BCAT1/2, BCKDHA/B, pBCKDHA, BCKDK, and PPM1K in Hep3B cells cultured under normal, glucose-starvation, or glutamine- starvation conditions for 48 h. (C) Western blot analysis of pBCKDHA expression in Hep3B cells when PPM1K was knocked down under glutamine-deprivation conditions. (D) Detection of BCKDHA and PPM1K knockdown efﬁciency in Hep3B stable cell lines under glutamine-starvation conditions. (E and F) Hep3B cells knocking down BCKDHA or PPM1K were labeled under glutamine-starvation conditions and measured by HRMS after 48 h. Percentages of 15N labeling in NEAAs and UMP (E). Percentages of 13C labeling in TCA intermediate metabolites (F).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER BT2 TargetMol Cat#T1483 TRIzol reagent Thermo Cat#15596-018 DNase Ambion Cat#AM1906 Western ECL Substrate Bio-Rad Cat#170561 Luna Omega Polar C18 LC column Phenomenex Cat#00D-4760-AN L-leucine(13C6 99%;15N,99%) CIL Cat#CNLM-281-H-0.05 L-Isoleucine(13C6 99%;15N,99%) CIL Cat#CNLM-561-H-0.05 L-Valine(13C6 99%;15N,99%) CIL Cat#CNLM-442-H-0.25 Polybrene Sigma Cat#H9268 Puromycin Sigma Cat#P8833 PEI Polysciences Cat#23966-2 Penicillin-streptomycin HyClone Cat#SV30010 RPMI-1640 medium Gibco Cat#31800-014 Dulbecco’s modified Eagle’s medium Thermo Fisher Scientific Cat#12800082 Certified Fetal Bovine Serum VivaCell, Shanghai, China Cat#C04001-500 Critical commercial assays Leu/Ile/Val Assay Kit Biovision Cat#K564-100 Apoptosis Detection Kit Vazyme Cat#A211-02 HiScript II 1st Strand cDNA Synthesis Kit Vazyme Cat#R211-02 AceQ qPCR SYBR Green Master Mix Vazyme Cat#Q111-03 EdU Apollo 643 In Vitro Kit RiboBio Cat#C10310-2 Matrigel BD Cat#356234 Deposited data WB raw data This manuscript; Mendeley Data https://doi.org/10.17632/gpp36hspvy.1 Experimental models: Cell lines Hep3B ATCC HB-8064; RRID:CVCL_0326 SNU-475 CoBioer Biosciences CBP60218; RRID:CVCL_0497

Techniques: Western Blot, Cell Culture, Expressing, Knockdown, Stable Transfection, Labeling

Figure 3. O-GlcNAcylation stabilizes PPM1K and facilitates dephosphorylation of BCKDHA consequentially under glutamine starvation conditions (A) Western blot analysis of PPM1K and pBCKDHA expression in Hep3B cells under normal, glucose, and glutamine conditions in the presence or absence of the proteasome inhibitor MG132. (B) Western blot analysis of O-GlcNAcylation levels in Hep3B cells under normal, glucose, and glutamine conditions. (C) Schematic diagram of O-GlcNAcylation cycles. (D) Western blot analysis of O-GlcNAc, PPM1K, and pBCKDHA when OGT (left panel) or MGEA5 was knocked down (right panel) in Hep3B cells under glutamine- starvation conditions.

Journal: Cell reports

Article Title: Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

doi: 10.1016/j.celrep.2022.111691

Figure Lengend Snippet: Figure 3. O-GlcNAcylation stabilizes PPM1K and facilitates dephosphorylation of BCKDHA consequentially under glutamine starvation conditions (A) Western blot analysis of PPM1K and pBCKDHA expression in Hep3B cells under normal, glucose, and glutamine conditions in the presence or absence of the proteasome inhibitor MG132. (B) Western blot analysis of O-GlcNAcylation levels in Hep3B cells under normal, glucose, and glutamine conditions. (C) Schematic diagram of O-GlcNAcylation cycles. (D) Western blot analysis of O-GlcNAc, PPM1K, and pBCKDHA when OGT (left panel) or MGEA5 was knocked down (right panel) in Hep3B cells under glutamine- starvation conditions.

Techniques: De-Phosphorylation Assay, Western Blot, Expressing

Figure 4. BCAA catabolism facilitates HCC cell-cycle progression under glutamine-starvation conditions (A) Evaluation of cell proliferation under different nutrient-deprivation conditions via an EdU assay. Hep3B cells were starved for 24 h before EdU treatment. Scale bars, 100 mm. (B) Statistical results of the experiments in (A) are shown in the bar graph. (C) Percentages of apoptotic cells under different nutrient-deprivation conditions. Hep3B cells were starved for 48 h. (D) Analysis of the cell cycle under the indicated nutrient-deprivation conditions. G1, S, and G2/M phases are indicated in blue, red, and green, respectively. Hep3B cells were starved for 48 h.

Journal: Cell reports

Article Title: Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

doi: 10.1016/j.celrep.2022.111691

Figure Lengend Snippet: Figure 4. BCAA catabolism facilitates HCC cell-cycle progression under glutamine-starvation conditions (A) Evaluation of cell proliferation under different nutrient-deprivation conditions via an EdU assay. Hep3B cells were starved for 24 h before EdU treatment. Scale bars, 100 mm. (B) Statistical results of the experiments in (A) are shown in the bar graph. (C) Percentages of apoptotic cells under different nutrient-deprivation conditions. Hep3B cells were starved for 48 h. (D) Analysis of the cell cycle under the indicated nutrient-deprivation conditions. G1, S, and G2/M phases are indicated in blue, red, and green, respectively. Hep3B cells were starved for 48 h.

Techniques: EdU Assay

Figure 5. Targeting both BCAA and glutamine metabolism dampens HCC progression in vivo (A) Hep3B cells stably expressing NTC, shBCKDHA, and shPPM1K were injected in BALB/c nude mice, combined with vehicle or BPTES treatment (n = 5 per group). Tumor growth was measured and plotted 16 days after inoculation. (B) Tumors in the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups were excised and compared at the end of the experiment. (C) Tumor weights were measured in the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. (D) Western blot analysis of BCKDHA, PPM1K, and pBCKDHA in tumor samples from the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. (E) BCAA levels in the serum of nude mice from NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. Three biological and technical replicates were used per group. (F) Hep3B cells stably expressing empty vector (EV), BCKDHA-S337A (S337A), and PPM1K were injected in BALB/c nude mice, combined with vehicle or BPTES treatment (n = 5 per group). Tumor growth was measured and plotted 16 days after inoculation. (G) Tumors in the EV + vehicle, EV + BPTES, BCKDHA-S337A (S337A) + BPTES, and PPM1K + BPTES groups were excised and compared at the end of the experiment. (H) Tumor weights were measured in the EV + vehicle, EV + BPTES, BCKDHA-S337A (S337A) + BPTES, and PPM1K + BPTES groups. (legend continued on next page)

Journal: Cell reports

Article Title: Branched-chain amino acid catabolism breaks glutamine addiction to sustain hepatocellular carcinoma progression.

doi: 10.1016/j.celrep.2022.111691

Figure Lengend Snippet: Figure 5. Targeting both BCAA and glutamine metabolism dampens HCC progression in vivo (A) Hep3B cells stably expressing NTC, shBCKDHA, and shPPM1K were injected in BALB/c nude mice, combined with vehicle or BPTES treatment (n = 5 per group). Tumor growth was measured and plotted 16 days after inoculation. (B) Tumors in the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups were excised and compared at the end of the experiment. (C) Tumor weights were measured in the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. (D) Western blot analysis of BCKDHA, PPM1K, and pBCKDHA in tumor samples from the NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. (E) BCAA levels in the serum of nude mice from NTC + vehicle, NTC + BPTES, shBCKDHA + BPTES, and shPPM1K + BPTES groups. Three biological and technical replicates were used per group. (F) Hep3B cells stably expressing empty vector (EV), BCKDHA-S337A (S337A), and PPM1K were injected in BALB/c nude mice, combined with vehicle or BPTES treatment (n = 5 per group). Tumor growth was measured and plotted 16 days after inoculation. (G) Tumors in the EV + vehicle, EV + BPTES, BCKDHA-S337A (S337A) + BPTES, and PPM1K + BPTES groups were excised and compared at the end of the experiment. (H) Tumor weights were measured in the EV + vehicle, EV + BPTES, BCKDHA-S337A (S337A) + BPTES, and PPM1K + BPTES groups. (legend continued on next page)

Techniques: In Vivo, Stable Transfection, Expressing, Injection, Western Blot, Plasmid Preparation

$Circ_CDR1as is a stable circRNA. ( A ) QRT-PCR assay for the relative expression of circ_CDR1as in THLE-2, Hep3B and Huh7 cells. ( B and C ) QRT-PCR assay for the relative expression of circ_CDR1as and GAPDH mRNA in Hep3B and Huh7 cells treated with RNase R or not. ( D and E ) QRT-PCR assay for the relative expression of circ_CDR1as and GAPDH mRNA in Hep3B and Huh7 cells disposed with Actinomycin D or not. ( F and G ) Nuclear-cytoplasmic fractionation assay for the relative expression of GAPDH, U6 and circ_CDR1as in the cytoplasmic and nuclear fractionations of Hep3B and Huh7 cells. * P < 0.05.$

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as is a stable circRNA. ( A ) QRT-PCR assay for the relative expression of circ_CDR1as in THLE-2, Hep3B and Huh7 cells. ( B and C ) QRT-PCR assay for the relative expression of circ_CDR1as and GAPDH mRNA in Hep3B and Huh7 cells treated with RNase R or not. ( D and E ) QRT-PCR assay for the relative expression of circ_CDR1as and GAPDH mRNA in Hep3B and Huh7 cells disposed with Actinomycin D or not. ( F and G ) Nuclear-cytoplasmic fractionation assay for the relative expression of GAPDH, U6 and circ_CDR1as in the cytoplasmic and nuclear fractionations of Hep3B and Huh7 cells. * P < 0.05.

Article Snippet: Human Liver Epithelial-2 (THLE-2, ATCC ® CRL-2706) and HCC cell line Hep3B (ATCC ® HB-8064) are purchased from American Type Culture Collection (ATCC; Manassas, VA, USA), HCC cell line Huh7 (BNCC337690) is bought from BeNa culture collection (Beijing, China).

Techniques: Quantitative RT-PCR, Expressing, Fractionation

Circ_CDR1as positively regulates HCC cell proliferation and metastasis. Hep3B and Huh7 cells are transfected with si-NC, si-circ_CDR1as, Vector or circ_CDR1as. ( A ) QRT-PCR assay for the relative expression of circ_CDR1as in transfected cells. ( B and E ) Colony formation assay for the number of the colonies formed by transfected cells. ( C, D, F and G ) MTT assay for the OD value of transfected cells at 490 nm. ( H – K ) Transwell assay for the migration and invasion abilities of transfected cells. ( L and M ) Western blot analysis for the protein levels of snail and E-cadherin in transfected cells. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as positively regulates HCC cell proliferation and metastasis. Hep3B and Huh7 cells are transfected with si-NC, si-circ_CDR1as, Vector or circ_CDR1as. ( A ) QRT-PCR assay for the relative expression of circ_CDR1as in transfected cells. ( B and E ) Colony formation assay for the number of the colonies formed by transfected cells. ( C, D, F and G ) MTT assay for the OD value of transfected cells at 490 nm. ( H – K ) Transwell assay for the migration and invasion abilities of transfected cells. ( L and M ) Western blot analysis for the protein levels of snail and E-cadherin in transfected cells. * P < 0.05.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Colony Assay, MTT Assay, Transwell Assay, Migration, Western Blot

Raf1 depletion inhibits HCC cell proliferation and metastasis. ( A and B ) QRT-PCR and Western blot assays for the relative expression of Raf1 in THLE-2, Hep3B and Huh7 cells. ( C – J ) Hep3B and Huh7 cells are introduced with si-NC or si-Raf1. ( C and D ) QRT-PCR and Western blot assays for the relative expression of Raf1 in treated cells. ( E ) Colony formation assay for the number of the colonies formed by treated cells. ( F and G ) MTT assay for the OD value of treated cells at 490 nm. ( H and I ) Transwell assay for the migration and invasion abilities of treated cells. ( J ) Western blot analysis for the protein levels of snail and E-cadherin in treated cells. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Raf1 depletion inhibits HCC cell proliferation and metastasis. ( A and B ) QRT-PCR and Western blot assays for the relative expression of Raf1 in THLE-2, Hep3B and Huh7 cells. ( C – J ) Hep3B and Huh7 cells are introduced with si-NC or si-Raf1. ( C and D ) QRT-PCR and Western blot assays for the relative expression of Raf1 in treated cells. ( E ) Colony formation assay for the number of the colonies formed by treated cells. ( F and G ) MTT assay for the OD value of treated cells at 490 nm. ( H and I ) Transwell assay for the migration and invasion abilities of treated cells. ( J ) Western blot analysis for the protein levels of snail and E-cadherin in treated cells. * P < 0.05.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Colony Assay, MTT Assay, Transwell Assay, Migration

Circ_CDR1as targets miR-1287, an upstream miRNA of Raf1. ( A ) The binding sites of miR-1287 on circ_CDR1as, as well as the mutant sites based on starBase. ( B and C ) Dual-luciferase reporter assay for the luciferase activities of circ_CDR1as-wt and circ_CDR1as-mut in Hep3B and Huh7 cells. ( D ) QRT-PCR assay for the relative expression of miR-1287 in Hep3B and Huh7 cells introduced with si-NC, si-circ_CDR1as, Vector or circ_CDR1as. ( E ) QRT-PCR assay the relative expression of miR-1287 in THLE-2, Hep3B and Huh7 cells. ( F ) QRT-PCR assay for the relative expression of miR-1287 in HCC tissues and adjacent normal tissues (n=105). ( G ) Pearson correlation analysis for the correlation between the levels of miR-1287 and circ_CDR1as in HCC tissues, r=−0.4748, P < 0.0001. ( H ) The binding sites of miR-1287 on the 3ʹUTR of Raf1, as well as the mutant sites based on starBase. ( I and J ) Dual-luciferase reporter assay for the luciferase activities of Raf1-wt and Raf1-mut in Hep3B and Huh7 cells. ( K – M ) Hep3B and Huh7 cells are transfected with miR-NC, miR-1287, anti-miR-NC or anti-miR-1287. ( K and L ) QRT-PCR assay the relative expression levels of miR-1287 and Raf1 in transfected cells. ( M ) Western blot analysis for the protein level of Raf1 in transfected cells. ( N ) Pearson correlation analysis for the correlation between the levels of miR-1287 and Raf1 in HCC tissues, r=−0.5270, P < 0.0001. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as targets miR-1287, an upstream miRNA of Raf1. ( A ) The binding sites of miR-1287 on circ_CDR1as, as well as the mutant sites based on starBase. ( B and C ) Dual-luciferase reporter assay for the luciferase activities of circ_CDR1as-wt and circ_CDR1as-mut in Hep3B and Huh7 cells. ( D ) QRT-PCR assay for the relative expression of miR-1287 in Hep3B and Huh7 cells introduced with si-NC, si-circ_CDR1as, Vector or circ_CDR1as. ( E ) QRT-PCR assay the relative expression of miR-1287 in THLE-2, Hep3B and Huh7 cells. ( F ) QRT-PCR assay for the relative expression of miR-1287 in HCC tissues and adjacent normal tissues (n=105). ( G ) Pearson correlation analysis for the correlation between the levels of miR-1287 and circ_CDR1as in HCC tissues, r=−0.4748, P < 0.0001. ( H ) The binding sites of miR-1287 on the 3ʹUTR of Raf1, as well as the mutant sites based on starBase. ( I and J ) Dual-luciferase reporter assay for the luciferase activities of Raf1-wt and Raf1-mut in Hep3B and Huh7 cells. ( K – M ) Hep3B and Huh7 cells are transfected with miR-NC, miR-1287, anti-miR-NC or anti-miR-1287. ( K and L ) QRT-PCR assay the relative expression levels of miR-1287 and Raf1 in transfected cells. ( M ) Western blot analysis for the protein level of Raf1 in transfected cells. ( N ) Pearson correlation analysis for the correlation between the levels of miR-1287 and Raf1 in HCC tissues, r=−0.5270, P < 0.0001. * P < 0.05.

Techniques: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Western Blot

Circ_CDR1as depletion represses HCC cell proliferation and metastasis by sponging miR-1287 to downregulate Raf1. Hep3B and Huh7 cells are introduced with si-NC, si-circ_CDR1as, si-circ_CDR1as+anti-miR-NC, si-circ_CDR1as+anti-miR-1287, si-circ_CDR1as+pcDNA or si-circ_CDR1as+Raf1. ( A and B ) QRT-PCR and Western blot assays for the relative expression of Raf1 in treated cells. ( C ) Colony formation assay for the number of the colonies formed by treated cells. ( D and E ) MTT assay for the OD value of treated cells at 490 nm. ( F and G ) Transwell assay for the migration and invasion abilities of treated cells. ( H ) Western blot analysis for the protein levels of snail and E-cadherin in treated cells. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as depletion represses HCC cell proliferation and metastasis by sponging miR-1287 to downregulate Raf1. Hep3B and Huh7 cells are introduced with si-NC, si-circ_CDR1as, si-circ_CDR1as+anti-miR-NC, si-circ_CDR1as+anti-miR-1287, si-circ_CDR1as+pcDNA or si-circ_CDR1as+Raf1. ( A and B ) QRT-PCR and Western blot assays for the relative expression of Raf1 in treated cells. ( C ) Colony formation assay for the number of the colonies formed by treated cells. ( D and E ) MTT assay for the OD value of treated cells at 490 nm. ( F and G ) Transwell assay for the migration and invasion abilities of treated cells. ( H ) Western blot analysis for the protein levels of snail and E-cadherin in treated cells. * P < 0.05.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Colony Assay, MTT Assay, Transwell Assay, Migration

Circ_CDR1as depletion inactivates MEK/ERK pathway by regulating miR-1287/Raf1 axis. Hep3B and Huh7 cells are introduced with si-NC, si-circ_CDR1as, si-circ_CDR1as+anti-miR-NC, si-circ_CDR1as+anti-miR-1287, si-circ_CDR1as+pcDNA or si-circ_CDR1as+Raf1. ( A and B ) Western blot analysis for the protein levels of MEK1, p-MEK1, ERKs and p-ERKs in treated cells. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as depletion inactivates MEK/ERK pathway by regulating miR-1287/Raf1 axis. Hep3B and Huh7 cells are introduced with si-NC, si-circ_CDR1as, si-circ_CDR1as+anti-miR-NC, si-circ_CDR1as+anti-miR-1287, si-circ_CDR1as+pcDNA or si-circ_CDR1as+Raf1. ( A and B ) Western blot analysis for the protein levels of MEK1, p-MEK1, ERKs and p-ERKs in treated cells. * P < 0.05.

Techniques: Western Blot

Circ_CDR1as depletion suppresses HCC tumor growth in vivo. Hep3B cells stably transfected with sh-circ_CDR1as or sh-NC are subcutaneously injected into BALB/c nude mice. ( A ) The volume of formed tumors. ( B ) The weight of formed tumors. ( C – E ) QRT-PCR assay for the relative expression of circ_CDR1as, miR-1287 and Raf1 in formed tumors. ( F ) Western blot analysis for the protein level of Raf1 in formed tumors. * P < 0.05.

Journal: Cancer Management and Research

Article Title: CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway

doi: 10.2147/CMAR.S252679

Figure Lengend Snippet: Circ_CDR1as depletion suppresses HCC tumor growth in vivo. Hep3B cells stably transfected with sh-circ_CDR1as or sh-NC are subcutaneously injected into BALB/c nude mice. ( A ) The volume of formed tumors. ( B ) The weight of formed tumors. ( C – E ) QRT-PCR assay for the relative expression of circ_CDR1as, miR-1287 and Raf1 in formed tumors. ( F ) Western blot analysis for the protein level of Raf1 in formed tumors. * P < 0.05.

Techniques: In Vivo, Stable Transfection, Transfection, Injection, Quantitative RT-PCR, Expressing, Western Blot

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cell lines hep3b atcc hb 8064 hepg2 atcc hb 8065 oligonucleotides primers (ATCC)

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