Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. Huh7, Hep3B and HepG2 liver cancer cells and immortalized human hepatocytes (THLE3) were incubated in the absence or in the presence of 5 ng/ml BMP9 in 0.1% FBS media and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.) are shown. B. THLE3 cells were incubated with different concentrations of BMP9 in 0.1% FBS media and counted after 4 days of treatment. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.) are shown. C. Proliferation curve of THLE3 cells incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS or in 10% FBS media. Data from one representative experiment (n = 3) out of 3 (mean ± S.D.) are shown. D. HepG2 cells were incubated with different concentrations of BMP9 in 0.1% FBS media and counted after 4 days of treatment. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). E. HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Data from 6 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t- test and data were compared to untreated samples, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. HepG2 cells were incubated for 1 hour with different concentrations of BMP9 in 0.1% FBS media. Western blots were performed with antibodies that recognized activated (phosphorylated) Smad1, 5 and 8 (P-Smad1,5,8) and Smad1 as loading control. A representative experiment of 2 is shown. B. HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Western blots were performed with antibodies that recognize Id1, P-Smad1,5,8 and total Smad1 (loading control). A representative experiment of 3 is shown. C. HepG2 stably expressing BRE-luciferase (HepG2-BRA) cells were plated and incubated with 0.1% FBS for 15 hours, and then treated with different concentrations of BMP9 for additional 15 hours. Luciferase activity was normalized to cell number. Data are shown as fold induction (relative to untreated cells) and are from one representative experiment (n = 4) out of 3 performed (mean ± S.D.). Statistical analysis was carried out using the paired t -test and data were compared to untreated samples, *** = P <0.001. D. HepG2 cells were incubated −/+ BMP9 (5 ng/ml) for different periods of time in 0.1% FBS media and Id1 levels were analyzed by qRT-PCR and normalized to 18S. Fold changes relative to untreated samples were determined (mean ± S.E.M, n = 3).

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Control, Stable Transfection, Expressing, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A, B and C. HepG2 cells were incubated for 1 hour with A. dorsomorphin (1 µM, Dm), B. LDN193189 (100 nM) or C. ALK1ecd (16 fold molar excess, F.M.E.) and −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Western blots were performed with antibodies that recognize P-Smad1,5,8 and Smad1 as loading control. A representative experiment of 2 is shown in each case. D. HepG2 cells were incubated as in A and counted at day 4. Data from 2 independent experiments performed in triplicate (mean ± S.E.M.). E. HepG2 cells were incubated as in B and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). F. HepG2 cells were incubated as in C and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). G. HepG2 cells were incubated without (C) or with dorsomorphin (Dm, 1 µM), LDN193189 (100 nM) or ALK1ecd (16 F.M.E) in 0.1% FBS media and counted at day 4. Data from at least 3 independent experiments performed in triplicate, displayed as percentage of C 0 samples (untreated cells, day = 0) (mean ± S.E.M). H. THLE3 cells were incubated with ALK1ecd (16 F.M.E) in 0.1% FBS media and counted at day 4. Data from 2 independent experiments performed in triplicate, displayed as percentage of C 0 (untreated cells, day = 0). Statistical analysis was carried out using paired t -test and data were compared to untreated samples, * = P <0.05, ** = P <0. 01, *** = P <0.001 or as indicated. n.s. = not significant.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Control

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. Independent stable cell lines expressing non-silencing (N.S.) and two different shRNAs targeted against BMP9 were generated by retroviral infection of HepG2 cells. BMP9 mRNA levels were determined by quantitative RT-PCR and normalized to 18S. Data expressed relative to N.S. cells (assigned an arbitrary value of 1) from 3 different experiments (mean ± S.E.M). B. Non-silencing (NS), shBMP9#1 and #2 stable HepG2 cell lines were incubated in 0.5% FBS and −/+ BMP9 (5 ng/ml) and counted at day 6. Data from 6 independent experiments performed in triplicate, displayed as percentage of N.S. untreated cells (mean ± S.E.M). C. HepG2 cells were plated in soft agar and treated with BMP9 (5 ng/ml) or with dorsomorphin (Dm, 1 µM) for 3 weeks (added twice a week) and the colony number was counted. Data (n = 4, BMP9; n = 8, Dm) are displayed as percentage of control cells (mean ± S.E.M). D. Previously generated non-silencing (N.S.), shBMP9#1 and #2 stable HepG2 cell lines were plated in soft agar and counted after 3 weeks. Data from 4 experiments, displayed as percentage of N.S. cells (mean ± S.E.M). Statistical analysis was carried out using paired t -test and data were compared to untreated N.S. or control samples or as indicated, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Generated, Retroviral, Infection, Quantitative RT-PCR, Incubation, Control

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. DNA synthesis as determined by thymidine incorporation in HepG2 cells cultured for 24 hours in the absence or presence of BMP9 (5 ng/ml). Data are mean ± S.E.M. of 4 independent experiments and are displayed as percentage of untreated cells. B. HepG2 cells were incubated with or without BMP9 (5 ng/ml) in 0.1% FBS media or in the presence on 10% FBS media for 24 hours and then nuclear DNA content was analyzed by flow cytometry. Percentages of cells corresponding to the different cell cycle phases are shown. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t -test and data were compared to untreated samples (0.1% FBS), * = P <0.05, ** = P <0. 01, *** = P <0.001. C, D, E. HepG2 cells were treated as in B for 4 days. C–D. Cells were trypsinized and C. Nuclear DNA content was analyzed by flow cytometry. Percentages of hypodiploid (apoptotic) cells are shown. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). D. Cells were incubated with annexin V and PI. Subsequently, fluorescence intensity was measured in a FACScan flow cytometer and the percentage of annexin V positive/PI negative cells was calculated. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). E. Apoptotic nuclei were visualized and counted after PI staining under a fluorescence microscope. A minimum of 1000 nuclei was counted per condition. Data from 2 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t -test and data were compared to 10% FBS media treated samples or as indicated, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: DNA Synthesis, Cell Culture, Incubation, Flow Cytometry, Fluorescence, Staining, Microscopy

Journal: JCI Insight

Article Title: Vasopressin mediates fructose-induced metabolic syndrome by activating the V1b receptor

doi: 10.1172/jci.insight.140848

Figure Lengend Snippet: ( A ) Transcriptional levels of the avpr1b in hypothalamus, pancreas, jejunum, kidney, liver, and spleen of WT mice on water Ctrl (clear purple bars) or receiving a 10% Frct solution for 30 weeks (solid purple bars). ( B ) Transcriptional levels of the avpr1a (red line) and the avpr1b (purple line) in liver of WT mice receiving a 10% Frct solution for 30 weeks. ( C and D ) Representative Western blot and densitometry ( n = 2 total blots) for the V1bR, fructokinase (KHK), and actin in human HepG2 cells Ctrl or exposed to AVP (250 nM), Frct (10 mM), or a combination of Frct plus AVP for 5 days. ( E ) KHK activity in HepG2 lysates from Ctrl, AVP, Frct, and Frct plus AVP cells. ( F ) Representative Western blot and densitometry ( n = 2 total blots) for V1bR and actin in HepG2 transduced with noncodifying shRNA ( scr ) or shRNA against avpr1b ( shAvpr1b ) at baseline or a Frct (10 mM) exposure. ( G ) Representative Western blot and densitometry ( n = 2 total blots) for KHK and actin in Ctrl, AVP, Frct, and Frct plus AVP HepG2 cells stably silenced for V1bR expression. ( H and I ) Representative Western blot ( n = 2 total blots) and densitometry for KHK, actin, and lipogenic enzymes FAS and ACC in the liver of WT and V1bR-KO mice on water Ctrl or receiving a 10% Frct solution for 30 weeks. The data in A and C–E are presented as the mean ± SD and analyzed by 1-way ANOVA with Tukey’s post hoc analysis. * P < 0.05, ** P < 0.01. For A and B and E , n = 6 mice per group. For C – E , n = 2 independent cultured plates. V1bR, vasopressin 1b receptor; avpr1b , vasopressin 1b receptor gene; avpr1a , vasopressin 1a receptor gene; KHK, ketohexokinase; Ctrl, control; AVP, vasopressin; Frct, fructose; scr , scramble; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase.

Article Snippet: V1b deletion in HepG2 cells was performed employing lentiviral particles containing either shRNA sequences specific for human V1bR (Santa Cruz Biotechnologies, sc-40277-v) or scramble — noncodifying — shRNA control (sc-108080).

Techniques: Western Blot, Activity Assay, Transduction, shRNA, Stable Transfection, Expressing, Cell Culture, Control

Journal: World Journal of Gastroenterology : WJG

Article Title: Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

doi: 10.3748/wjg.v9.i12.2732

Figure Lengend Snippet: Identification of CYP2E1 mRNA expression in HepG2-CYP2E1 and HepG2 cells by RT-PCR with beta-actin as inter-nal control. Lane 1: Markers (λ/EcoR I and Hind III), 2: RT-PCR products of HepG2 cells showing only beta-actin 462 bp, 3: RT-PCR products of HepG2-CYP2E1 cells showing 462 bp of beta-actin and 690 bp of CYP2E1.

Article Snippet: Yoshitomi et al[ 17 ] have established in HepG2 cells stable expression of a series of human CYP subtypes, such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: World Journal of Gastroenterology : WJG

Article Title: Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

doi: 10.3748/wjg.v9.i12.2732

Figure Lengend Snippet: Cytotoxicity of NDEA against HepG2-CYP2E1 and HepG2 cells. Cells were exposed to various concentrations of NDEA. Relative survival rate was represented as the relative toxicity to the control culture without NDEA. The results presented were the average of six duplications (-x ± s). aP < 0.05, bP < 0.01 vs HepG2 cells.

Article Snippet: Yoshitomi et al[ 17 ] have established in HepG2 cells stable expression of a series of human CYP subtypes, such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

Techniques: Control

Journal: World Journal of Gastroenterology : WJG

Article Title: Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

doi: 10.3748/wjg.v9.i12.2732

Figure Lengend Snippet: MN rates in HepG2-CYP2E1 and HepG2 cells in-duced by NDEA. Cells were exposed to various concentrations of NDEA. The data were expressed as per thousand of binucleated cells with MN. aP < 0.05, bP < 0.01 vs HepG2 cells.

Article Snippet: Yoshitomi et al[ 17 ] have established in HepG2 cells stable expression of a series of human CYP subtypes, such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

Techniques:

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Diethyl maleate stimulates Nrf2 but not FOXO-dependent gene expression. HepG2 cells were exposed to DEM (1 mM) for 4 h or 16 h in serum-free DMEM (A) , followed by analysis of G6PC and SELENOP mRNA levels by qRT-PCR. Data are means of five independent experiments + SD. (B) HepG2 cells, grown to approx. 75% confluence, were held on serum-free medium for 18 h, followed by exposure to DEM at the given concentrations for 2 h. If indicated, this was followed by a 30 min incubation with insulin (100 nM). Nuclear extracts were prepared and binding of endogenous FOXO1 to an oligonucleotide containing FOXO binding elements analyzed in an ELISA-based approach. Data are means of three independent experiments + SD. (C) HepG2 cells were exposed to DEM for 4 h or 16 h, followed by analysis of Nrf2 target gene mRNA levels by qRT-PCR. Data are means of five independent experiments + SD. Statistical significance was assessed using Student's t -test: *P < 0.05, **P < 0.01 vs. respective control.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Gene Expression, Quantitative RT-PCR, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Role of FOXO1 cysteine residues and of FOXO phosphorylation in DEM response. (A) HEK293 or HepG2 cells transiently expressing GFP-coupled human mutant FOXO1 [FOXO1(C#1-7S)] were exposed to the given concentrations of DEM followed by analysis of subcellular distribution of GFP-FOXO1 as described in the legend to A. (B) HepG2 cells were transfected with a plasmid encoding a GFP-coupled form of FOXO1(WT) or the cysteine-deficient mutant FOXO1(C#1-7S). Cells were exposed to DEM (3 mM) or DMSO (solvent control) for 2 h, followed by 30 min exposure to insulin (100 nM) as indicated. FOXO1 and phospho-FOXO1 were detected by Western blotting, actin detection was used as gel loading control. Blots are representative of three independent experiments. (C) DEM stimulates JNK. HepG2 cells were incubated in the presence of 10 µM SP600125 (or DMSO as solvent control) for 1 h, followed by exposure to DEM at the given concentration for 1 h in the continued presence of SP600125 (or solvent control). Cells were lysed and subjected to SDS-PAGE and Western analysis of JNK and cJun phosphorylation. The blots shown are representative of two independent experiments. (D) DEM-induced nuclear accumulation of FOXO1 is independent of JNK. HepG2 cells were transfected with a plasmid expressing GFP-FOXO1 24 h prior to exposure to SP600125 (10 µM) and DEM (3 mM) as in (C). Analysis of subcellular localization of GFP-FOXO1 was done by categorizing cells according to the predominant FOXO1 distribution into “cytoplasm”, “cytoplasm/nucleus”, and “nucleus”. Subcellular distribution of GFP-FOXO1 in cells was calculated from three independent experiments. In each of these experiments, at least 198 cells were categorized per setting. Data are given as means + SD.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Solvent, Control, Western Blot, Incubation, Concentration Assay, SDS Page

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Analysis of subcellular localization of FOXO1 upon exposure to Leptomycin B or DEM. HepG2 cells were transfected with an expression plasmid coding for GFP-FOXO1, followed by exposure to the given compounds and analysis of subcellular distribution of GFP-FOXO1 as described before. (A) Cells were exposed to Leptomycin B (LMB) at 30 ng/mL for 30 min, followed by addition of insulin (100 nM) for another 30 min. In the panel on the right, insulin was added first (30 min), followed by LMB (30 min) prior to analysis of GFP-FOXO1 subcellular localization. (B) Cells were treated as in (A), with DEM (10 mM) instead of LMB. Subcellular distribution of EGFP-FOXO1 in cells was calculated from three independent experiments. On average, 210 (min: 124, max: 267) cells were categorized per condition in each of the independent experiments. Data are given as means + SD. (C) HepG2 cells expressing EGFP-FOXO1 were exposed to insulin (causing nuclear exclusion) and DEM (3 mM) or solvent control (DMSO) for the indicated times. Analysis of subcellular distribution of EGFP-FOXO1 was done as described in Materials and Methods.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Transfection, Expressing, Plasmid Preparation, Solvent, Control

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Analysis of subcellular localization of FOXO1 upon exposure to stressful stimuli. HepG2 cells were transfected with an expression plasmid coding for GFP-FOXO1, followed by exposure to the given compounds and analysis of subcellular distribution of GFP-FOXO1 as described before. (A) Cells were treated with DEM (2 mM) for 30 min, followed by addition of insulin (left), sodium arsenite (100 µM, middle) or copper sulfate (10 µM, right) for another 60 min. Data are means of at least three independent experiments + SD. On average, 149 (min: 92, max: 240) cells were categorized per condition in each of the independent experiments. (B) Schematic summary of findings: DEM causes nuclear accumulation of FOXO1 by blocking its nuclear exclusion. Nuclear exclusion elicited by stimuli such as insulin, copper ions or arsenite is attenuated by DEM. Abbreviations in scheme: InsR, insulin receptor; IGF1-R, insulin-like growth factor.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Transfection, Expressing, Plasmid Preparation, Blocking Assay

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Glutathione depletion and oxidation in HepG2 cells exposed to diethyl maleate. Analysis of glutathione (GSH, A) and glutathione disulfide (GSSG, B) content of hepatoma cell lysates following exposure to the given concentrations of DEM or solvent (DMSO, “0 mM DEM”) for 120 min. Data are means of four independent experiments + SD.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Solvent

Journal: Redox Biology

Article Title: Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

doi: 10.1016/j.redox.2018.09.010

Figure Lengend Snippet: Diethyl maleate causes nuclear accumulation of FOXO1. (A, B) HEK293 or HepG2 cells transiently expressing GFP-coupled human FOXO1 (wildtype) were exposed to the given concentrations of DEM (“0”: solvent control, DMSO) for 30 min, followed by analysis of subcellular distribution of GFP-FOXO1. At least 200 cells were categorized for each independent experiment with respect to the predominant subcellular localization of GFP-FOXO1. Data are presented as means of three independent experiments + SD. (C) Images of HepG2 cells expressing GFP-FOXO1 prior to (Ctrl.) and after addition of insulin (Ins, 100 nM) and DEM (3 mM) as described in . The images shown provide examples of cells with GFP-FOXO1 in both nuclear and cytoplasmic compartments (Ctrl), predominantly cytoplasmic (insulin) and predominantly nuclear (DEM) localization. (D) Age-synchronized L1 larvae of the C. elegans TJ356 strain stably expressing a DAF-16::GFP fusion protein were transferred to NGM agar plates supplemented with DEM at 1 mM. 0.1% DMSO was used as control (Ctrl); exposure was for 24 h. Examples of worms with predominantly cytoplasmic (left) and nuclear localization (right, see arrows, inset) of DAF-16::GFP are shown. The experiment was performed at least three independent times. Bar = 100 µm.

Article Snippet: HepG2 human hepatoma cells and HEK293 human embryonic kidney cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Expressing, Solvent, Control, Stable Transfection

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Metabolism of Beclomethasone Dipropionate by Cytochrome P450 3A Enzymes

doi: 10.1124/jpet.112.202556

Figure Lengend Snippet: (A) Quantification of BDP metabolites produced by A549 cells treated with BDP or BDP with esterase inhibitors (+EI). (B) Relative quantification of BDP metabolites produced by DPX2 cells treated with BDP or BDP with esterase inhibitors (+EI). Data are the mean and standard deviation from six replicates. n.d. Signifies that the metabolite was not detected. (C and D) CYP3A enzyme mRNA abundance, measured by qPCR in A549 (C) and DPX2 (D) cells. Data are represented as the number of mRNA copies per 10,000 copies of β2-macroglobulin (a “housekeeping” gene). Statistics used for A549 cell data analysis were one-way analysis of variance with Dunnett’s post-hoc test. For DPX2 cell data analysis two-way ANOVA with Bonferronni post-hoc testing was used. Data are the mean and standard deviation from 6 replicates. n.d. Signifies that mRNA was not detected. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Article Snippet: DPX2 cells (HepG2 background with human PXR stably overexpressed to drive the expression of a CYP3A4 reporter gene construct by PXR agonists) were provided by Dr. Judy Raucy (Puracyp Inc., Carlsbad, CA).

Techniques: Produced, Quantitative Proteomics, Standard Deviation

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Excess glucose availability promotes GLUT1 clearance from the plasma membrane (A) HeLa cells were cultured for 24 h in media with no glucose, then switched to high glucose media (25 mM) for the indicated amount of time. Biotin-labeling was performed at the post-glucose shift time points. Following biotinylation, labeled cells were lysed and surface proteins were affinity purified with NeutrAvidin beads (Thermo Scientific). Analysis was performed by SDS-PAGE and immunoblot with antibodies that recognize GLUT1, Na + /K + ATPase, and GAPDH. (B,C) Quantification of captured GLUT1 (B) and Na + /K + ATPase (C) for the experiment shown in (A) was performed over multiple biological replicates (n ≥ 3). GLUT1 measurements were taken of the whole lane using FIJI. Immunoblots for biological replicate experiments are provided in . (D) HeLa cells stably expressing mCherry-CaaX (red) were cultured in no glucose media for 24 h (top row) then shifted to high glucose (25 mM) for 24 h (bottom row), at which point the samples were fixed for immunofluorescence detection with GLUT1 antibody (green). Zoomed images provided in the far right column correspond to the yellow dashed-line inset boxes in the "MERGE" image to the left. (E) Quantification of co-localization shown in (D) was measured by Pearson correlation on Softworx software (n = 30 cells), p = 3.75 × 10 -8 . (F) HeLa cells stably expressing GLUT1-GFP (green) were cultured using the conditions indicated in (D). Prior to imaging, cells were pulse-labeled with FM4-64 (red), a lipophilic tracer dye that inserts into the outer leaflet of the cell membrane. Live cells were incubated on ice in 8 μM cold FM4-64 for ∼5 min before imaging. Zoomed images provided in the far right column correspond to the yellow dashed-line inset boxes in the "MERGE" image to the left. (G) Quantification of the results shown in (F). Pearson correlation coefficient was measured using Softworx software (n = 30 cells), p = 1.69 × 10 -24 . For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Clinical Proteomics, Membrane, Cell Culture, Labeling, Affinity Purification, SDS Page, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Software, Imaging, Incubation

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Glucose-stimulated clearance of GLUT1 results in trafficking to lysosomes (A) Imaging of endogenous GLUT1 was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with GLUT1 antibody (green) and LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization (as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 1 × 10 -5 . (B) Imaging of stably expressed GLUT1-GFP (green) was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 1 × 10 -6 . (C) Imaging of stably expressed GLUT1-FLAG, which harbors a FLAG tag on its first exofacial loop, was performed in HeLa cells cultured in no glucose media for 24 h then switched to high glucose media (25 mM) for the indicated time and fixed. Samples were imaged via immunofluorescence and probed with FLAG antibody (green) and LAMP1 antibody (red). A schematic of the detection strategy is shown in the top left of the panel. Quantification of co-localization as measured by Pearson correlation on Softworx software (n = 30 cells) is shown in the graph at the top right of the panel. ∗∗ indicates p < 0.004. For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Imaging, Cell Culture, Immunofluorescence, Software, Stable Transfection, FLAG-tag

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Characterization of the GLUT1 trafficking itinerary stimulated by excess glucose availability HeLa cells expressing the three versions of GLUT1 described in Figure 2 were cultured in media lacking glucose for 24 h then either fixed or switched to high glucose media and fixed at the indicated time points. Cells were then probed by immunofluorescence for the endosomal proteins VPS35 (A-B) or CD63 (C-D) (red). In each case, co-localization was analyzed for endogenous GLUT1 (left, green), GLUT1-GFP (middle, green), or GLUT1-FLAG (right, green). Co-localization of GLUT1 signal with VPS35 (B) and CD63 (D) was quantified over the glucose stimulation time course. Co-localization measurements were made in Softworx using Pearson correlation coefficient (n = 30 cells). ∗∗ indicates p < 0.002. (E) Summarized profile of the GLUT1 trafficking itinerary stimulated by excess glucose availability. Heat maps showing co-localization of endogenous GLUT1 (top), GLUT1-GFP (middle), and exofacial GLUT1-FLAG (bottom) with different markers along the endocytic/endosomal trafficking route. For each time point and each marker, at least 21 measurements were made of the Pearson coefficient of correlation using Softworx software. The color in each box is weighted based on the average Pearson coefficient (n ≥ 21) at the indicated time point. (F) HeLa cells harboring a doxycycline-inducible dominant-negative VPS4 variant (VPS4 E228Q -HA) were cultured in no glucose media + 1 μg/ml doxycycline for 24 h then fixed or switched to high glucose media + doxycycline and fixed at the indicated time point. Cells were then imaged for immunofluorescence detection of HA (red) and GLUT1 (green). VPS4 E228Q is a dominant-negative mutant that accumulates on late-endosomal compartments responsible for sorting cargo into intraluminal vesicles. GLUT1 puncta that co-localize with, and are surrounded by, VPS4 E228Q -HA are marked with white arrows. (G) Quantification of the experiments represented in (F) by measuring the Pearson coefficient of correlation (n = 30 cells) using Softworx software. ∗∗ indicates p < 0.001. (H) HeLa cells stably expressing both GLUT1-GFP (green) and doxycycline-inducible VPS4 E228Q -HA were cultured as described in (F) and then imaged for immunofluorescence detection of HA (red). GLUT1-GFP puncta that co-localize with, and are surrounded by, VPS4 E228Q -HA are marked with white arrows. (I) Quantification of the experiments represented in (H) by measuring the Pearson coefficient of correlation (n = 30 cells) using Softworx software. ∗∗ indicates p < 0.02. For all experiments, p values were computed using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Expressing, Cell Culture, Immunofluorescence, Marker, Software, Dominant Negative Mutation, Variant Assay, Stable Transfection

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: The clathrin-binding motif and PY motifs of TXNIP are required for glucose-mediated GLUT1 trafficking to lysosomes (A) HeLa cells stably expressing a doxycycline-inducible expression vector were cultured using the conditions described in Figure 1 D and, for the induced samples, additionally treated with 1 μg/ml doxycycline for the last 24 h before fixation. Cells were fixed and imaged for immunofluorescence detection of GLUT1 (green) and LAMP1 (red), a marker of lysosomal compartments. Zoomed images provided in the bottom row correspond to the blue dashed-line inset boxes of the top row. (B) Quantification of the experiments represented in panel (A) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition shown. (C) HeLa cells and txnip knockout equivalents (clone 2) stably expressing GLUT1-GFP (green) were cultured as indicated in (A) then fixed for immunofluorescence detection of LAMP1 (red), a marker of lysosomal compartments. (D) Quantification of the experiments represented in panel (C) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition shown. (E) Schematic representation of TXNIP illustrating the predicted arrestin fold domain (yellow), the clathrin-binding motif (orange), and the two PY motifs (green). (F) Complementation analysis of HeLa cells stably expressing GLUT1-GFP (green) with the txnip gene knocked out via CRISPR/Cas9. The knockout cells were stably transfected with either an empty vector or a vector expressing wild-type TXNIP, a clathrin-binding mutant ( cb ), or a py motif mutant ( py ) expressed from a doxycycline-inducible promoter. Cells were cultured as indicated in Figure 1 D with doxycycline added the last 24 h to induce expression of the indicated protein. Cells were fixed and imaged by immunofluorescence for detection of LAMP1 (red), a marker of lysosomal compartments. (G) Quantification of the results shown in panel (F) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) for each condition indicated. The dashed line and area shaded in red indicate the average Pearson’s coefficient and standard deviation (respectively) for the condition in which there is no glucose and TXNIP expression is not induced. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM. All measurements of Pearson coefficient of correlation were performed using Softworx software.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Binding Assay, Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunofluorescence, Marker, Knock-Out, CRISPR, Transfection, Mutagenesis, Standard Deviation, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: TXNIP is dispensable for GLUT1 ubiquitin modification (A) HeLa cells stably expressing GLUT1-GFP were stably transfected with either empty vector (pINDUCER20) or vector expressing wild type, clathrin-binding mutant ( cb ), or py motif mutant (py) TXNIP under the control of Tet-on gene expression system. 1 μg/ml doxycycline was added to induce expression of TXNIP for 24 h prior to collection of cell lysate. Cell lysates were incubated with recombinant WWP1-FLAG at 4 ° C overnight then WWP1-FLAG was pulled down using αFLAG magnetic beads. Elution was performed using FLAG peptide. Lysates and eluates were resolved by SDS-PAGE and analyzed by immunoblot. Immunoblotting of GAPDH was performed as a loading control. (B) HeLa cells stably expressing GLUT1-GFP were transiently transfected with either a wild type or py mutant TXNIP-FLAG expression plasmid. When cells reached 100% confluence, they were collected in lysis buffer and incubated with αFLAG magnetic beads for 1 h at 4 ° C with rotation. TXNIP-FLAG was eluted with FLAG peptide and samples were resolved by SDS-PAGE then analyzed by immunoblot. GAPDH was used as a loading control. (C) HeLa cells stably expressing GLUT1-GFP and a dox-inducible clathrin-binding mutant TXNIP were transiently transfected with either wild-type WWP1-FLAG or a mutant WWP1-FLAG with all 4 ww domains mutated. TXNIP CB was induced with 1 μg/ml doxycycline 24 h before collecting lysates. Cells were then collected in lysis buffer and lysates were incubated with αFLAG magnetic beads for 1 h at 4 ° C with rotation. WWP1 was eluted using FLAG peptide and samples were resolved by SDS-PAGE and analyzed by immunoblot. GAPDH was used as a loading control. (D) HEK293T cells stably expressing FLAG-Ub were split into either 1) regular 25 mM glucose DMEM media, 2) DMEM media with no glucose, or 3) no glucose DMEM media and switched to 25 mM glucose media 2 h before collection. All cells were transiently transfected with a GLUT1-GFP expression plasmid. When cells reached 100% confluency, sample 3 cells were switched to high glucose (25 mM) DMEM media and lysates were collected 2 h later then incubated with magnetic FLAG affinity beads for 1 h at 4 ° C with rotation. FLAG-Ub was eluted using FLAG peptide; samples were resolved by SDS-PAGE, and analyzed by immunoblot. α-Tubulin was used as a loading control. (E) Quantification of the eluate GLUT1 signal for three biological replicates (n = 3) of the experiment shown in (D). ∗∗ indicates a significant difference (p < 0.05) compared to the no glucose condition (lane 2). (F) txnip knockout HeLa cells stably expressing constitutive GLUT1-FLAG and a dox-inducible TXNIP expression plasmid were transiently transfected with HA-Ub. 24 hours before collecting lysates, cells were either mock-treated (sample 2) or treated with 1 µg/ml doxycycline (sample 3) to induce TXNIP expression. As a control, HeLa cells with a stably integrated empty vector (i.e., endogenous TXNIP but no GLUT1-FLAG expression) were also analyzed (sample 1). Lysates were incubated with magnetic αFLAG affinity beads for 1 h at 4°C with rotation and eluted using FLAG peptide. Samples were resolved by SDS-PAGE and analyzed by immunoblot. GAPDH was used as a loading control. (G) Quantification of the eluate HA-Ub signal in four biological replicates (n = 4) of the experiment shown in (F). (H) HeLa cells stably expressing constitutive GLUT1-FLAG and dox-inducible TXNIP vectors were transiently transfected with either empty vector, HA-Ub, and/or WWP1 as indicated in the figure. 24 hours after inducing TXNIP with 1 μg/ml doxycyxline, cells were collected and lysed. Lysates were incubated with magnetic αFLAG affinity beads for 1 h at 4 ° C with rotation. GLUT1-FLAG was eluted with FLAG peptide and samples were resolved by SDS-PAGE then analyzed by immunoblot. GAPDH was used as a loading control. (I) Quantification of HA-Ub signal for at least three biological replicates (n ≥ 3) of the experiments shown in (H). Double asterisk (∗∗) indicates a significant difference (p < 0.05) compared to the empty vector control. All p-values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Ubiquitin Proteomics, Modification, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Control, Gene Expression, Incubation, Recombinant, Magnetic Beads, SDS Page, Western Blot, Lysis, Knock-Out

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: Mapping of cytosolic lysines required for lysosomal trafficking of GLUT1 (A) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R) were cultured in media lacking glucose for 24 h then cultured for another 24 h in fresh media lacking glucose (“no glucose”) or shifted to fresh media with high glucose (25 mM) for 24 h (“high glucose”) prior to fixation and imaging for immunofluorescence detection of LAMP1 (red). Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (B) Quantification of the results shown in (A) was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. ∗∗ indicates p < 1x10 -5 . (C) Schematic of GLUT1 illustrating the primary amino acid sequence of N-terminal and C-terminal cytosolic tails. Lysine residues in the N-terminal and C-terminal cytosolic tails are highlighted in red. A similar schematic illustrating the lysine residues in the major cytosolic loop is shown in Figure S11 B. (D) HeLa cells stably expressing either wild-type GLUT1-GFP, GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R), GLUT1-GFP with the 6 lysines on the major cytosolic loop mutated to arginine (6K loop →R ), or GLUT1-GFP with the 5 cytosolic lysines outside of the major loop mutated to arginine (5K tails →R) were cultured as in (A) prior to fixation and imaging for immunofluorescence detection of LAMP1 (red). Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (E) Quantification of the results shown in panel D was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. The dashed line and area shaded in red indicate the average Pearson’s coefficient and standard deviation for the WT GLUT1-GFP co-localization with LAMP1 under glucose-starved conditions. All measurements of Pearson coefficient of correlation were performed using Softworx software. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Stable Transfection, Expressing, Cell Culture, Imaging, Immunofluorescence, Sequencing, Standard Deviation, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet: TXNIP-mediated trafficking of GLUT1 requires its cytosolic lysine residues (A) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R) were stably transfected with a doxycycline-inducible vector harboring wild-type TXNIP. Cells were cultured as indicated in Figure 1 D prior to fixation and imaging for immunofluorescence detection of LAMP1 (red), a marker of lysosomal compartments. Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (B) Quantification of the results shown in panel A was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. (C) HeLa cells stably expressing either wild-type GLUT1-GFP or GLUT1-GFP with all cytosolic lysines mutated to arginine (11K cyto →R GLUT1) were stably transfected with a doxycyclin-inducible vector harboring wild-type TXNIP. Cells were cultured as indicated in Figure 1 D. Prior to imaging, cells were placed on ice and switched to cold (4°C) buffer containing the lipophilic tracer dye FM4-64 (8 μM) (red) in order to label the plasma membrane. Live cells were imaged in cold buffer immediately to ensure retention of FM4-64 at the plasma membrane. Zoomed images in the bottom row correspond to the blue dashed line inset boxes of the image above. (D) Quantification of the results shown in panel C was performed by measuring the Pearson coefficient of correlation for 30 cells (n = 30) with each condition indicated. All p values were measured using a two sample Student’s t-Test in Microsoft Excel. A P value < 0.05 was considered statistically significant and is indicated by ∗∗. Data are represented as mean +/- SEM. All measurements of Pearson coefficient of correlation were performed using Softworx software.

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Culture, Imaging, Immunofluorescence, Marker, Clinical Proteomics, Membrane, Software

Journal: iScience

Article Title: Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

doi: 10.1016/j.isci.2023.106150

Figure Lengend Snippet:

Article Snippet: GLUT1 antibody , Proteintech , Proteintech Cat# 21829-1-AP, RRID: AB_10837075.

Techniques: Purification, Produced, Recombinant, Infection, Stable Transfection, Expressing, Diagnostic Assay, CRISPR, Plasmid Preparation, Software