Journal: BioMed Research International

Article Title: Hydroxytyrosol Plays Antiatherosclerotic Effects through Regulating Lipid Metabolism via Inhibiting the p38 Signal Pathway

doi: 10.1155/2020/5036572

Figure Lengend Snippet: Effects of HT and selective inhibitor of p38 on lipid metabolism and inflammation pathway molecules. HepG2 cells treated with diverse concentrations (10, 25, and 50 μ M) of HT or SB203580 (20 μ M) for 12 h were subjected to CCK-8 assay and Western blot analysis. The data was present as means ± SD, n = 3 for each group. ∗ p < 0.05, ∗∗ p < 0.01 vs. the control condition. NC: negative control, namely, solvent vehicle control (DMSO). NS: not significant vs. DMSO. SB: SB203580.

Article Snippet: Human hepatocarcinoma HepG2 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: CCK-8 Assay, Western Blot, Control, Negative Control, Solvent

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism

doi: 10.1152/ajpgi.00322.2010

Figure Lengend Snippet: Nrf2 induction of SHP gene expression. A: semiquantitative PCR analysis of gene expression using total RNA isolated from primary hepatocytes of 2-mo-old male wild-type (+, lane 1) and Nrf2−/− (−, lane 2) mice, or from Nrf2−/− hepatocytes transfected with a pcDNA3 control (pc, lane 3) or a Nrf2 expression plasmid (Nrf2, lane 4) that expresses a full length mouse Nrf2. B: real-time PCR analysis of gene expression in primary hepatocytes of 2-mo-old male wild-type mice overexpressed with control green fluorescent protein (GFP; GFPAd) or Nrf2 adenovirus (Nrf2Ad). Nqo1, a Nrf2 target gene, was used as a positive control. C: real-time PCR analysis of gene expression in primary hepatocytes of 2-mo-old male wild-type mice treated with oxidized free fatty acid DHA (oxDHA, 50 μm, 5 h). D: real-time PCR analysis of gene expression in mouse hepatocyte Nmuli cells treated with 0.1% butylated hydroxyanisole (BHA; dissolved in DMSO) for 5 h. E: real-time PCR analysis of liver gene expression in 2-mo-old male wild-type mice fed with 0.5% BHA for 2 wk (n = 5/group). F: real-time PCR analysis of gene expression in Nrf2+/+ and Nrf2−/− hepatocytes treated with 0.1% BHA for 5 h. G: real-time PCR analysis of liver gene expression in 2-mo-old male Nrf2+/+ and Nrf2−/− mice fed with 0.5% BHA for 2 wk. B–F used HPRT and F–G used β-actin as an internal control. H: Nrf2−/− hepatocytes were transduced with GFP control or SHP adenovirus and the total TG and cholesterol (Chol) contents were determined. I: luciferase (Luc.)/β-galactosidase (β-gal, gal.) activities (act.) determined in HEK293 cells transfected with the SHP promoter reporter (∼2 kb) in the presence of Nrf2 (200 ng) expression plasmid and its coactivators (200 ng each). The β-gal expression vector (100 ng) was cotransfected as a control for transfection efficiency. Luciferase activity was normalized against β-gal activity. J: chromatin immunoprecipitation (ChIP) assays of Nrf2 coimmunoprecipitation (CoIP) on the SHP promoter region containing a conserved antioxidant response element (ARE) by using specific Nrf2 antibodies. Nrf2 expression vector was cotransfected with the mouse SHP promoter in HepG2 cells for 2 days and the cells were harvested. Rabbit anti-Nrf2 antibody (5 μg) was used for the CoIP. Rabbit IgG was used as a negative control. The fragment targeting for the ARE was amplified by PCR. The primers designed at the distal site of SHP promoter (−5 kb) was set as a negative control. The location of ARE and primers used for ChIP assays is presented in Fig. S2. Data (B–F) are represented as means ± SE of triplicate assays. Ad, adenovirus; *P < 0.01.

Article Snippet: For chromatin immunoprecipitation (ChIP) assays, HepG2 cells were used: chromatin was cross-linked and immunoprecipitated with rabbit anti-Nrf2 antibodies (sc-13032, Santa Cruz) and rabbit normal IgG (serving as a negative control).

Techniques: Gene Expression, Isolation, Transfection, Control, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control, Transduction, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Amplification

Journal: Journal of King Saud University - Science

Article Title: Cytotoxic potential of Commicarpus plumbagineus extracts against liver cancer cell lines through In-Vitro and In-Silico methods

doi: 10.1016/j.jksus.2024.103253

Figure Lengend Snippet: Fig. 2. The cytotoxicity of the F2 extract from C. plumbagineus was evaluated on human liver cancer cells (HepG2 and HuH7) and non-cancerous cells (Huvec) using various concentrations (0–700 µg/mL) for a 24-hour treatment. Cell viability was assessed through the MTT assay, and statistical analysis was carried out utilising Student’s t-test. The data, presented as the mean ± stan dard deviation, were derived from three replicates. Significance was established at *p < 0.05 in comparison to the control group.

Article Snippet: Human hepatocellular carcinoma cells HuH7 and HepG2 and human umbilical vein endothelial non-cancerous cell line (HUVECs) (DSMZ, Germany) were maintained in DMEM (Gibco) supplemented with fetal R. Alghamdi et al. Journal of King Saud University - Science 36 (2024) 103253 bovine serum (FBS 10 %) (Gibco), 10 mM L-glutamine, at 37 ◦C in CO2 incubator.

Techniques: MTT Assay, Derivative Assay, Comparison, Control

Journal: Journal of King Saud University - Science

Article Title: Cytotoxic potential of Commicarpus plumbagineus extracts against liver cancer cell lines through In-Vitro and In-Silico methods

doi: 10.1016/j.jksus.2024.103253

Figure Lengend Snippet: Fig. 3. Fluorescent photomicrographs depict the HepG2 cancer cell line stained with DAPI after exposure to the F2 extract (300 μg/mL). The panels illustrate the following: (A) Untreated HepG2 cell line, (B) Treated HepG2 cell line after 24 h of incubation.

Article Snippet: Human hepatocellular carcinoma cells HuH7 and HepG2 and human umbilical vein endothelial non-cancerous cell line (HUVECs) (DSMZ, Germany) were maintained in DMEM (Gibco) supplemented with fetal R. Alghamdi et al. Journal of King Saud University - Science 36 (2024) 103253 bovine serum (FBS 10 %) (Gibco), 10 mM L-glutamine, at 37 ◦C in CO2 incubator.

Techniques: Staining, Incubation

Journal: Journal of King Saud University - Science

Article Title: Cytotoxic potential of Commicarpus plumbagineus extracts against liver cancer cell lines through In-Vitro and In-Silico methods

doi: 10.1016/j.jksus.2024.103253

Figure Lengend Snippet: Fig. 4. Acridine orange/ethidium bromide staining of HepG2 cells to detect apoptosis induced by F2 extract from C. plumbagineus (300 µg/mL). (A) negative control (B) treated cells. Live cells are uniformly green (L). In contrast, apoptotic cells (AP) are characterised by green and fragmented chromatin.

Article Snippet: Human hepatocellular carcinoma cells HuH7 and HepG2 and human umbilical vein endothelial non-cancerous cell line (HUVECs) (DSMZ, Germany) were maintained in DMEM (Gibco) supplemented with fetal R. Alghamdi et al. Journal of King Saud University - Science 36 (2024) 103253 bovine serum (FBS 10 %) (Gibco), 10 mM L-glutamine, at 37 ◦C in CO2 incubator.

Techniques: Staining, Negative Control

Journal: Journal of King Saud University - Science

Article Title: Cytotoxic potential of Commicarpus plumbagineus extracts against liver cancer cell lines through In-Vitro and In-Silico methods

doi: 10.1016/j.jksus.2024.103253

Figure Lengend Snippet: Fig. 5. The effects of the F2 extract from C. plumbagineus on the migration of HepG2 cells were evaluated. Figure A displays images of the wound monolayer of HepG2 cells at two points: immediately after wounding (t = 0 h) and after a 24-hour incubation period. Cells were either left untreated (control) or treated with the extract at 87.5 µg/mL. Figure B illustrates the calculated cell migration rate using the methodology described in the materials and methods section. The experiments were performed in triplicate, and statistical analysis was conducted using Student’s t-test (*p < 0.05 vs control).

Article Snippet: Human hepatocellular carcinoma cells HuH7 and HepG2 and human umbilical vein endothelial non-cancerous cell line (HUVECs) (DSMZ, Germany) were maintained in DMEM (Gibco) supplemented with fetal R. Alghamdi et al. Journal of King Saud University - Science 36 (2024) 103253 bovine serum (FBS 10 %) (Gibco), 10 mM L-glutamine, at 37 ◦C in CO2 incubator.

Techniques: Migration, Incubation, Control

Journal: Biochemical pharmacology

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

doi: 10.1016/j.bcp.2023.115615

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

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

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

Journal: Biochemical pharmacology

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

doi: 10.1016/j.bcp.2023.115615

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

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

Techniques: Cell Cycle Assay, Expressing, Western Blot

Journal: Biochemical pharmacology

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

doi: 10.1016/j.bcp.2023.115615

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

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

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

Journal: Biochemical pharmacology

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

doi: 10.1016/j.bcp.2023.115615

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

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

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Biochemical pharmacology

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

doi: 10.1016/j.bcp.2023.115615

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

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

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

Journal: 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

Journal: Cancer Science

Article Title: Histone methyltransferase SUV420H1 / KMT5B contributes to poor prognosis in hepatocellular carcinoma

doi: 10.1111/cas.16038

Figure Lengend Snippet: Pathways associated with SUV420H1 using Gene Ontology (GO) analysis. (A) GO terms involving SUV420H1 in Huh‐7 cell line that were derived neither from hepatitis B virus (HBV) nor C virus (HCV) (nBnC) hepatocellular carcinoma (HCC). GO enriched in siSUV420H1‐transfected cells, compared with siEGFP‐transfected cells, are indicated. (B) GO terms involving SUV420H1 in SNU475 cell line of HBV‐derived HCC. Gene ontologies enriched in siSUV420H1‐transfected cells, compared with siEGFP‐transfected cells, are indicated. (C) Common GO terms in Huh‐7 and SNU475 cell lines.

Article Snippet: Commercially available SUV420H1 CRISPR/Cas9 knockout (KO) plasmid (sc‐404615; Santa Cruz Biotechnology), SUV420H1 homology‐directed repair (HDR) plasmid (sc‐404615‐HDR; Santa Cruz Biotechnology), and Control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) as negative control were transfected to HCC cells (Huh‐7 and HepG2) with UltraCruz transfection reagent (sc‐395739; Santa Cruz Biotechnology) following the manufacturer's protocol, and then subjected to selection by 1 μg/mL puromycin (sc‐108071; Santa Cruz Biotechnology) for 24 h prior to qRT‐PCR, immunofluorescence, and injection into mice.

Techniques: Derivative Assay, Virus, Transfection

Journal: Cancer Science

Article Title: Histone methyltransferase SUV420H1 / KMT5B contributes to poor prognosis in hepatocellular carcinoma

doi: 10.1111/cas.16038

Figure Lengend Snippet: In vivo examination employing xenograft hepatocellular carcinoma (HCC) tumors using CRISPR/Cas9 specific for SUV420H1 in Huh‐7. (A) Immunofluorescence for SUV420H1 and HDR genes cotransfected into Huh‐7 cell nucleus. (B) The relative mRNA expression level in CRISPR/Cas9 control and CRISPR/Cas9 SUV420H1 KO. (C) The changeover time of tumor progression in CRISPR/Cas9 control and CRISPR/Cas9 SUV420H1 KO. (D) Photographs of subcutaneous tumor and macroscopic specimens on day 28. Scale bars, 30 μm. DAPI, diamidino‐2‐phenylindole; GFP, green fluorescent protein; HDR, homology‐directed repair. Results are presented as mean ± SEM. * p < 0.05, *** p < 0.001. All the experiments were done in triplicate.

Article Snippet: Commercially available SUV420H1 CRISPR/Cas9 knockout (KO) plasmid (sc‐404615; Santa Cruz Biotechnology), SUV420H1 homology‐directed repair (HDR) plasmid (sc‐404615‐HDR; Santa Cruz Biotechnology), and Control CRISPR/Cas9 plasmid (sc‐418922; Santa Cruz Biotechnology) as negative control were transfected to HCC cells (Huh‐7 and HepG2) with UltraCruz transfection reagent (sc‐395739; Santa Cruz Biotechnology) following the manufacturer's protocol, and then subjected to selection by 1 μg/mL puromycin (sc‐108071; Santa Cruz Biotechnology) for 24 h prior to qRT‐PCR, immunofluorescence, and injection into mice.

Techniques: In Vivo, CRISPR, Immunofluorescence, Expressing, Control

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 binds to KD2 of tissue factor pathway inhibitor (TFPI). (A) TFPI expression vector was transfected into HEK 293 cells. Binding of Kunitz‐2 domain (KD2) of TFPI with MG1113 was then confirmed using immunoprecipitation (IP). A band was observed by western blot (WB) only in cases of TFPI constructs possessing KD2. Mock: human embryonic kidney (HEK) 293 cells were transfected without TFPI expression vector. (B) Two perpendicular views are shown with three polypeptides in different colors. Immunoglobulin (Ig) heavy chain is denoted by VH and CH. Ig kappa light chain is indicated by Vk and Ck. Mapping of epitopes and paratopes, defined as residues within the intersubunit distance of 4.5 Å, on surface representations of KD2 and Fab of MG1113. Left, epitopes on KD2 are shown in three different colors. Orange, yellow, and red indicate putative activated factor X (FXa)‐binding residues, which overlap with Fab of MG1113‐binding residues of KD2. Arg107 (in red) is a key residue in the inhibition of FXa. Right, paratopes on MG1113 are shown in magenta for VH residues and in pink for Vk residues. (C) MG1113 no longer binds to the construct from which the epitope of TFPI is removed. Ab, antibody; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Construct, Residue, Inhibition

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: Neutralizing effect of MG1113 on the function of tissue factor pathway inhibitor‐α (TFPI‐α). (A) The activity of 1 nM activated factor X (FXa) is reduced by 10 nM TFPI. The activity of FXa is recovered (n = 3) after treatment with MG1113 (0.625‐40 nM). (B) Activation of 10 nM factor X (FX) by extrinsic FXase, comprised of 10.5 pM tissue factor (TF) and 0.5 nM activated factor VII (FVIIa), is inhibited by 9 nM TFPI. Generation and activity of FXa are recovered (n = 4) after treatment with MG1113 (0.13‐800 nM). (C) After factor VIII (FVIII) deficient plasma is treated with MG1113 through thrombin generation assay, with increasing concentration, thrombin generation is also increased (n = 3). (D, E) In a modified prothrombin time (mPT) assay, when FVIII or factor IX (FIX) deficient plasma is treated with MG1113, with increasing concentration, clotting time is further shortened (n = 3). The graph represents mean and standard deviation

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Activity Assay, Activation Assay, Clinical Proteomics, Concentration Assay, Modification, Coagulation, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 not only binds to tissue factor pathway inhibitor‐β (TFPI‐β) but also neutralizes it. (A) Binding of human umbilical vein endothelial cells (HUVECs) and MG1113 is confirmed. Differently from human IgG (hIgG), which is a negative control, with increasing concentration of MG1113 (0.003 ‐ 200 nM), increased binding of MG1113 to HUVECs (n = 3) is observed. (B) The factor X (FX) is activated by activated factor VII (FVIIa) and tissue factor, which is expressed on stimulated HUVECs by tumor necrosis factor‐α. With increasing concentration of MG1113 (0.006‐500 nM), generation of activated factor X (FXa) is increased (n = 2). The graph represents mean and standard deviation

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Binding Assay, Negative Control, Concentration Assay, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 restores blood loss and clotting time of a hemophilia A (HA)‐induced rabbit. (A) Rabbits received saline or factor VIII (FVIII) neutralizing antibody (Nab) (10 mg/kg) 45 minutes before bleeding induction. Rabbits received saline or MG1113 (0.1, 0.25, 1, 5 mg/kg) 35 min before bleeding induction. Bleeding was observed for 1 hour. Hemoglobin level was reduced by MG1113 treatment in a concentration‐dependent manner. (B) Activated partial thromboplastin time (aPTT) was delayed after treatment with FVIII NAb, but not changed after MG1113 treatment. (C) Modified prothrombin time (mPT) was shortened by MG1113 treatment. (D) Free tissue factor pathway inhibitor (TFPI) level was not changed by FVIII neutralizing antibody (NAb) treatment. However, it was reduced by MG1113 treatment. The graph represents mean and standard deviation. In the test group of 0.25 mg/kg, at which MG1113 started to affect, free TFPI level remained at approximately 20%‐22% of that of the group not treated with MG1113. Each dot represents an entity (n = 8‐15). Outliers were excluded from the data set (Grubb’s test, P < .01). The group not treated with MG1113 and other groups were analyzed using one‐way analysis of variance, assuming a normal distribution. The significance level was set at P < .05. § Two‐stage linear step‐up procedure; † Games‐Howell pairwise comparison; ‡ Dunnett post hoc analysis

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Coagulation, Saline, Concentration Assay, Modification, Standard Deviation, Comparison

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: Confirmation for the ability of MG1113 in an HA‐induced rabbit through subcutaneous administration. The same concentration of MG1113 was administered intravenously or subcutaneously. Blood loss, activated partial thromboplastin time (aPTT), modified prothrombin time (mPT), and free tissue factor pathway inhibitor (TFPI) levels were then measured. (A) Hemoglobin level, (C) mPT, and (D) the level of free TFPI are reduced by MG1113 treatment through subcutaneous administration. (B) aPTT is delayed by FVIII neutralizing antibody treatment, but not changed by MG1113 treatment. The graph represents mean and standard deviation. Each dot represents an entity (n = 6‐15). The group not treated with MG1113 and other groups were analyzed using one‐way analysis of variance assuming a normal distribution. The significance level was set at P < .05. † Games‐Howell pairwise comparison; ‡ Dunnett post hoc analysis

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Concentration Assay, Modification, Standard Deviation, Comparison

Journal: Cancer Communications

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

doi: 10.1002/cac2.12600

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

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

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

Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside inhibited the proliferation and colony formation of Hep3B and SNU-449 cells and the restoration by CAXII overexpression. Relative inhibition rates of Hep3B, SNU-449 and also THLE-3 in response to different concentrations of Tiliroside were calculated by comparing the OD value of NC, at 24, 48, 72, and 96 h, respectively ( A – C ). The representative colony formation of Hep3B and SNU-449 cells intervened by 40 μM Tiliroside and NC ( H ). The relative colony formation efficiency showed significant reduction of colony 10 days after Tiliroside intervention in both Hep3B and SNU-449 cells ( F ). The relative CAXII expression was significantly higher in CAXII-transfection group than non-transfection group in both Hep3B and SNU-449 cell lines ( D ). The relative CAXII expression was significantly decreased in CAXII-KO group than none transfection group in both Hep3B and SNU-449 cell lines ( E ). The inhibition rates brought by Tiliroside were significantly increased in CAXII overexpression group compared to both non-transfection group and empty vector group, at 48 and 72 h, respectviely, in Hep3B and SNU-449 cells. On the contrary, The inhibition rates were significantly decreased in CAXII-KO group compared to both non-transfection group and none-KO group, at 24, 48 and 72 h, respectviely in both cell lines ( E ). Data are presented as the mean ± standard deviation (SD); NS not significant, NC negative control.

Article Snippet: Human hepatocellular carcinoma cell Hep3B, SNU-449 and liver normal cell THLE-3 were purchased from American Type Culture Collection (ATCC, USA) and characterized by identity verification with STR analysis 2019.

Techniques: Over Expression, Inhibition, Expressing, Transfection, Plasmid Preparation, Standard Deviation, Negative Control

Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside inhibited the migration and invasion abilities of Hep3B and SNU-449. The wound healing assay was made in Hep3B ( A ) and SNU-449 ( B ) treated by 40 μM Tiliroside or NC at 0, 24, 48 h, respectively. The bar graphics present the percentage of wound recovery in Hep3B ( C ) and SNU-449 ( D ). Cell wounds healed significantly faster in NC groups than those in Tiliroside group. Transwell chamber invasion assay for Hep3B and SNU-449 cells intervened by Tiliroside , and NC. Images were taken after 24 h or 12 h incubation. The average of cell number was counted by 3 randomly chosen different fields in both Hep3B and SNU-449 cells ( E , F ). Values represent the mean ± SD. NS non-significant, NC negative control.

Article Snippet: Human hepatocellular carcinoma cell Hep3B, SNU-449 and liver normal cell THLE-3 were purchased from American Type Culture Collection (ATCC, USA) and characterized by identity verification with STR analysis 2019.

Techniques: Migration, Wound Healing Assay, Invasion Assay, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside restrained the 3D formation ability and CD133 expression of Hep3B and SNU-449 cells. The representative 3D spheroid models of Hep3B ( A ) and SNU-449 ( B ) cells treated by 40 μM Tiliroside and NC. The relative cross-section area formation efficiency showed significant reduction of area of Hep3B ( C ) and SNU-449 ( D ) in Tiliroside groups. Relative inhibition rates of Hep3B and SNU-449 in response to Tiliroside were calculated by comparing the fluorescence value of Tiliroside to NC, at 24, 48, 72, 96 and 120 h, respectively ( E ). Tiliroside (40 μM) treatment significantly reduced the CD133 relative expression levels in both 3D cultured Hep3B and SNU-449 cells (P < 0.001) ( F ). NC negative control.

Article Snippet: Human hepatocellular carcinoma cell Hep3B, SNU-449 and liver normal cell THLE-3 were purchased from American Type Culture Collection (ATCC, USA) and characterized by identity verification with STR analysis 2019.

Techniques: Expressing, Inhibition, Fluorescence, Cell Culture, Negative Control

Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside targeted CAXII enzyme as U-104 did and reduced its quantity and activity. Tiliroside also modulated the expression of E2F1 , E2F3 whose relative expressions were higher in Hep3B and SNU-449 than in THLE-3. CAXII was predicted to be the potential targets of Tiliroside by Swiss target prediction tool ( A ). In both 2D and 3D culture systems, Tiliroside has significantly decreased the relative concentration of CAXII compared to NC group in both Hep3B, SNU-449 and THLE-3 at 24 h, 48 h and 72 h respectively ( B ). Dose-dependent inhibition of CAXII esterase activity by Tiliroside showed the IC50s were 47.54 ± 3.6 μM and 34.67 ± 2.7 μM for Hep3B and SNU-449, respectively ( C ), and that for U-104 were 6.3 ± 3.6 μM in Hep3B and 4.78 ± 3.6 μM in SNU-449 ( D ) (The concentration showed x axis stands for 0.5, 1.5, 5,10,20,80 μM, respectively). The relative expression levels of E2F1 and E2F3 were significantly higher in Hep3B and SNU-449 compared to that in THLE-3 ( E ). The relative expression levels of E2F1 ( F ) and E2F3 ( G ) in Hep3B, SNU-449 and THLE-3 cells were significantly downregulated by 40 μM Tiliroside in both 2D and 3D culture systems. Data are presented as the mean ± standard deviation (SD); NS not significant, NC negative control.

Article Snippet: Human hepatocellular carcinoma cell Hep3B, SNU-449 and liver normal cell THLE-3 were purchased from American Type Culture Collection (ATCC, USA) and characterized by identity verification with STR analysis 2019.

Techniques: Activity Assay, Expressing, Concentration Assay, Inhibition, Standard Deviation, Negative Control

Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside elevated the activity of Caspase-3. After 48 h intervention of 40 μM Tiliroside or DMSO, the absorbance was significant higher in the Tiliroside group than NC, apoptosis inhibited NC and apoptosis inhibited Tiliroside group in Hep3B ( A ) and SNU-449 ( B ), cultured in either 2D or 3D systems, respectively. CASP3-specific activities were also significantly higher in Tiliroside treated group compared to NC in Hep3B, SNU-449 and THLE-3 cells ( C ); NC negative control, NS non-significant.

Article Snippet: Human hepatocellular carcinoma cell Hep3B, SNU-449 and liver normal cell THLE-3 were purchased from American Type Culture Collection (ATCC, USA) and characterized by identity verification with STR analysis 2019.

Techniques: Activity Assay, Cell Culture, Negative Control

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on hepatic apoC-III expression. Rat primary hepatocytes were transduced with Foxo1 or LacZ vector at an MOI of 50 PFU/cell or mock-transduced with PBS. After 24 hours of transduction, the intracellular levels of apoC-III (A), Foxo1 (B), and GK (C) mRNA were determined by real-time RT-PCR using β-actin mRNA as control. The effect of Foxo1 on hepatic apoC-III expression in response to insulin was assayed in HepG2 cells. Cells were transduced with Foxo1, Foxo1-ADA, or control LacZ vector (50 PFU/cell) in the absence or presence of insulin at different concentrations. Twenty-four hours after transduction, cells were collected for determination of the intracellular levels of apoC-III mRNA induced by Foxo1 (D) and Foxo1-ADA (E). *P < 0.05, **P < 0.005; significantly different from controls. NS, not significant by ANOVA. Data were from 3 independent experiments.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Expressing, Transduction, Plasmid Preparation, Quantitative RT-PCR

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on the human APOC3 promoter activity. (A) The APOC-III promoter–directed luciferase reporter system. The wild-type and mutant IRE sequences are underlined. (B) Foxo1-mediated induction of the APOC3 promoter activity. HepG2 cells were transfected by pHD317 together with Foxo1 construct, or with both Foxo1 and Foxo1-Ø256 constructs. For each construct, 1 μg of DNA for each construct was used in transfection. For normalization of transfection efficiency, 1 μg pCMV5-LacZ DNA was included for normalization of transfection efficiency. (C) The APOC3 promoter variants in the luciferase reporter system. (D) Responses of APOC3 promoter variants to Foxo1 production. HepG2 cells were transfected with individual test plasmids in the absence (–) or presence (+) of pCMV5-Foxo1. The relative luciferase activity, after normalizing to β-gal activity, was compared between basal (–) and Foxo1-inducible (+) conditions. (E) Responses of wild-type and mutant APOC3 promoters to insulin. Test plasmids were transduced into HepG2 cells in the presence and absence of pCMV5-Foxo1 transfection in culture media, either supplemented with or without insulin (30 nM). The relative luciferase activity in transduced cells was determined using β-gal activity as control. *P < 0.001 vs. controls.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Activity Assay, Luciferase, Mutagenesis, Transfection, Construct

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Molecular interaction between Foxo1 and the APOC3 promoter. Molecular association between Foxo1 and the APOC3 promoter was analyzed by EMSA and ChIP. Aliquots of Foxo1 protein from linked in vitro transcription-translation products (5 μg) were incubated with 2.5 μl of radioactively labeled DNA corresponding to –467/–440 nt in the human APOC3 promoter (WT-IRE) (A), a mutant APOC3 IRE (mt-IRE) containing 2 substitutions, of –A458C and –A460G, and a control PEPCK IRE DNA (B), followed by electrophoresis through 8% nondenaturing polyacrylamide gels for 30 minutes. Lane 1, DNA probe alone. Lane 2, DNA probe + Foxo1 protein lysates. Lane 3, DNA probe + Foxo1 protein lysates + anti-Foxo1 antibody (1 μg). Lane 4, DNA probe + Foxo1 protein lysates + nonlabeled competitor DNA at a molar concentration of 50-fold excess. Free, shifted, and supershifted DNA bands were visualized by autoradiography. For ChIP assay, HepG2 cells were transduced with Foxo1 vector at an MOI of 50 PFU/cell. Cells were harvested 24 hours later and subjected to ChIP using PBS as a negative control (lane 5), control IgG (lane 6), and anti-Foxo1 antibody (lane 7). The coimmunoprecipitated chromatin DNA was analyzed by immunoblot (C) using anti-Foxo1 antibody and PCR (D) using the primers that correspond to –655/–20 nt of the APOC3 promoter.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: In Vitro, Incubation, Labeling, Mutagenesis, Electrophoresis, Concentration Assay, Autoradiography, Transduction, Plasmid Preparation, Negative Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: Fetuin-A (A, D), FGF23 (B, E) and merge (C, F) expression visualized respectively by Fitc and rhodamine IS in primary hepatocytes and in HepG2. Arrows show Fetuin-A immunopositivity not co-localized. The negative Ctrl exhibits only nuclear DAPI staining (C, F in blue). Scale Bars: 50μm.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: Fetuin-A (A), FGF23 (B), Clathrin (C) and MERGE expression (D) visualized respectively by Fitc, CY5 and rhodamine IS in HepG2. Scale Bars: 50μm.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: mRNA expression of Fetuin-A and FGF23 in primary hepatocytes and HepG2 (A). qRT PCR of FGF23 in primary hepatocytes compared to podocytes (negative control) and OS (Positive Ctrl) (B). Asterisks indicate significant differences: ***=p<0.001 by Student's t-test. n=3/group.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Expressing, Quantitative RT-PCR, Negative Control

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: WB of FGF23 on HepG2 FGF23/IP (AI) and on the portion non-FGF23/IP (negative Ctrl) with FGF23 (AII). WB of Fetuin-A on FGF23/IP (AIII) and on the aliquot non- FGF23/IP (AIV). Cofilin (bottom of the gel) acted as loading control (LC). Cell localization of Fetuin-A and FGF23 interactions detected by Duolink in situ experiment in HepG2 (B) and negative Ctrl (C) Scale bars: A, B: 50µm.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: In Situ

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: Cells Cultured medium harvested from primary hepatocytes, HepG2, and OS for measurement of intact FGF23 release, assessed by ELISA. (n=3/group; mean ±SD; data were normalized by Janus Green Nuclear marker; ***=p<0.001 OS vs all the other groups).

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Marker

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: Changes in FGF23, Fetuin-A and TNFα mRNA expression after 24h from the addition of scalar FGF23 dose (A, B, C). qRT PCR of FGF23, Fetuin-A and TNFα in HepG2 after 24h from FGF23 overexpression (D, E, F). Asterisks indicate significant differences versus HepG2 (ctrl): *=p<0.01, **=p<0.01, ***=p<0.001 by Student's t-test. n=3/group.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: International Journal of Biological Sciences

Article Title: FGF23 and Fetuin-A Interaction in the Liver and in the Circulation

doi: 10.7150/ijbs.23256

Figure Lengend Snippet: Semi-quantitative PCR of Fetuin-A promoter of HepG2 after FGF23/CHIP. Product size of human Fetuin-A promoter 146bp (A). Changes in Fetuin-A promoter expression after 24h from the addition of scalar FGF23 dose and after 24h from FGF23 overexpression (B). Asterisks indicate significant differences versus HepG2 (ctrl): **=p<0.01, ***=p<0.001 by Student's t-test. n=3/group.

Article Snippet: 20000 cells/cm 2 of HepG2 were transfected with 10nM human ORFclone cDNA clone FGF23 (Myc-DDK tagged) (OriGene Technologies Inc.MD, USA) using Lipofectamine2000 (Invitrogen) as transfection agent for 24h.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression