Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Western blot immunoreactivity of the polyclonal anti-MPV17 antibody Ab 93374 ( left ) and the monoclonal anti- human MPV17 antibodies 6F5 and 5D2 on extracts of human and murine cells of different MPV17 genotypes. Left extracts from human U2OS cells, untransfected or transfected with the MPV17 expression clone SC118652 (origene), were probed with the antibody ab 93374 (abcam). Center untransfected U2OS cells with MPV17 +/+ endogenous genotype probed with the monoclonal antibodies 6F5 and 5D2. Right Murine embryo fibroblast (MEF) cells of MPV17 +/+ or MPV17 -/- genotype probed with the monoclonal antibodies 6F5 and 5D2. Indicated marker sizes were transferred from Ponceau stained marker bands run in parallel. Loading was controlled as indicated by reaction of the filters with an anti-ß-actin antibody. 10 μg of protein were separated on SDS gels and analysed by Western blot according to ( left panel ), and Kinkley et al. respectively

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Western Blot, Transfection, Expressing, Marker, Staining

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Ab93374 (Abcam) recognizes MPV17 specific structures in U2OS cells transiently transfected with an MPV17 expression construct in immunofluorescence analysis. Ab93374 signal ( green ) is largely missing in nontransfected cells. The immunoreactivity does colocalise with peroxisomal (anti-catalase, mouse polyclonal, Abcam ab88650) and mitochondrial (anti-complex IV mitochondrial subunit I, mouse monoclonal, Invitrogen 459600) markers ( red ). Immunofluorescence (IF) method was according to Pircher et al. using a MicroRadiance confocal scanning system (Bio-Rad) in combination with a Zeiss Axiophot microscope. Colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Transfection, Expressing, Construct, Immunofluorescence, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: In U2OS cells, 5D2 antibody detects a punctate pattern colocalising with a peroxisomal (PMP70) but not with a mitochondrial (MitoTracker) marker. Top Rabbit anti PMP70 antibody ( decorated green ) was a gift from W. Just, Heidelberg. Secondary antibodies: Alexa Fluor 555 anti-mouse and Alexa Fluor 488 anti-rabbit. Bottom Mitotracker 7510 (Invitrogen) was used as recommended by the supplier. Secondary antibody: Alexa 488 anti-mouse. IF method and microscopy according to Kinkley et al. using a Zeiss LSM confocal microscope. Mathematical colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: 5D2 colocalisation with a marker of early endosomes (EEA1) and lysosomes (LAMP1) in U2OS cells. Top Partial colocalisation of 5D2 with EEA1 (rabbit anti-human). Bottom colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig.

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Colocalisation of 5D2 and LAMP1 mediated immunofluorescence in a U2OS single cell. IF and analysis of 5D2 and Colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig. . In addition a merge of the two immunofluorescence pictures is depicted

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Immunofluorescence

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 1. Endogenous 14‑3‑3β expression in osteosarcoma tissues and in matched normal tumor‑adjacent bone tissues from patients. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. *P<0.05. (B) 14‑3‑3β protein expression levels were assessed by western blotting. All data are presented as mean ± standard deviation.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 2. Endogenous 14‑3‑3β expression in osteosarcoma cell lines. Endogenous expression of 14‑3‑3β was assessed in the osteosarcoma cell lines U2OS, Saos‑2 and MG63 and in the human normal osteoblast cell line hFOB1.19. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. (B) 14‑3‑3β protein expression levels were assessed by western blotting. (C) Quantification of protein expression relative to β‑actin (loading control). All data are presented as mean ± standard deviation. *P<0.05 vs. hFOB1.9.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Control, Standard Deviation

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 3. 14‑3‑3β knockdown in MG63 osteosarcoma cells. MG63 cells were transfected with either a 14‑3‑3β‑specific siRNA (knockdown group) or a non‑specific control siRNA (control group). Untransfected cells were used as the blank group. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. (B) 14‑3‑3β protein expression levels were assessed by western blotting. (C) Quantification of protein expression relative to β‑actin (loading control). All experiments were repeated three times with three replicates each. All data are presented as mean ± standard deviation. *and #P<0.05 knockdown group vs. blank group and control group, respectively.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Transfection, Control, Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Oxidative stress increases OGT and OGA expression, OGA activity, and O-GlcNAc levels. U2OS cells were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 10, unless otherwise indicated. A, expression of OGT, OGA, and actin, as well as O-GlcNAc levels, was assessed in NETN lysates (5 μg) by Western blotting (WB). Protein load was assessed by total protein stain (colloidal Coomassie G-250) and by Western blotting (actin). Molecular mass (MW) markers are indicated. B, quantitation of actin normalized to total protein (G-250). C, quantitation of O-GlcNAc levels normalized to actin. D, quantitation of OGT expression normalized to actin. E, NETN lysates (5 μg) were assayed for OGT activity using [3H]UDP-GlcNAc (0.5 μCi) and CKII acceptor peptide (1 mm). n = 4, three technical replicates per assay. F, quantitation of OGA expression normalized to actin. G, NETN lysates (5 μg) were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 6, two technical replicates per assay. B–G, data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Dunnett's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Expressing, Activity Assay, Western Blot, Staining, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA fusion proteins express, maintain catalytic activity, and biotinylate proteins in vivo. A, schematic of the OGA-mBirA fusion proteins. CD and AD represent the catalytic (β-N-acetylglucosaminidase) domain and acetyltransferase-like domain of OGA, respectively. B–D, U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with or without biotin (25 μm, 16 h) or TMG (100 nm, 20 h) as indicated. Proteins were extracted in TCL buffer. B, equal amounts of protein (10 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: OGA, HA, Myc, and actin. n = 3. C, desalted lysates were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 3, representative data from one experiment is shown. Error bars indicate the intra-assay standard deviation from two technical replicates. D, equal amounts of protein (4.5 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 2. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Activity Assay, In Vivo, Transfection, SDS Page, Western Blot, Intra Assay, Standard Deviation, Migration

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA fusion proteins localize to and biotinylate proteins in the nucleus, cytoplasm, and mitochondria of U2OS cells. U2OS cells were transfected with pcDNA3.1 (A), OGA-mBirA-HA (B–D), or Myc-mBirA-OGA (E–G), treated with (A, C, D, F, and G) or without (B and E) biotin (25 μm, 16 h), and treated with (D and G) or without (A–C, E, and F) H2O2 (2.5 mm, 2 h). Cells were fixed, permeabilized, and stained for BirA and biotin. Nuclei and mitochondria were stained with Hoechst 33342 and MitoTracker Orange CMTMRos, respectively. White triangles indicate co-localization (orange) of MitoTracker and OGA (B and E). Images were acquired at ×63 magnification on a Zeiss Axio Examiner 710NLO-Meta multiphoton microscope. n = 3. Scale bar, 15 μm.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Transfection, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA-HA and Myc-mBirA-OGA biotinylate proximal proteins differentially in response to oxidative stress. U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with biotin (25 μm, 16 h) in the presence or absence of H2O2 (2.5 mm, 2 h). A, equal amounts of protein (5 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 4. B, densitometric total lane profiles for each lane from the biotin signal in A. Asterisks are used to highlight a subset of the biotinylated signals that are altered by oxidative stress. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Transfection, Lysis, SDS Page, Western Blot, Migration

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: SILAC-BioID-MS/MS strategy used to identify the basal and oxidative stress-dependent interactome of OGA. A, U2OS cells were labeled with light, medium, or heavy isotopes of arginine and lysine for six generations. In experiment 1, cells were transfected with pcDNA3.1 (heavy) or OGA-mBirA-HA (light, medium), treated with biotin (25 μm, 16 h), and treated with vehicle (medium, heavy) or H2O2 (light; 2.5 mm, 2 h, n = 1). In experiment 2, Myc-mBirA-OGA was transfected in replacement of OGA-mBirA-HA. For each experiment, proteins were extracted in denaturing TCL buffer and combined in equal amounts. The biotinylated proteins were isolated on NeutrAvidin-agarose in denaturing conditions, eluted in 2% (w/v) SDS (95 °C), and precipitated with acetone. Peptides were generated by trypsin and LysC digestion, separated by basic reversed phase (bRP) fractionation, and identified by mass spectrometry (LC-ESI-MS/MS). Subsequently, protein-protein interactions were validated by co- IP and Western blotting. B, for the SILAC experiments (n = 1, each), equal amounts of protein (10 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, OGA, HA, Myc, and actin. Protein load was assessed by total protein stain (Sypro Ruby) and by Western blotting (actin). Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Multiplex sample analysis, Tandem Mass Spectroscopy, Labeling, Transfection, Isolation, Generated, Fractionation, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Western Blot, Lysis, SDS Page, Staining, Migration

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Oxidative stress induces the association of OGA with FAS, FLNA, HSC70, and OGT. U2OS cells were treated with Vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 3. A, anti-OGA antibody (IP: OGA; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous OGA from NETN cell lysates (500 μg), of which 1.5–2% (input) and 30–40% (immunoprecipitate) were analyzed by SDS-PAGE. OGA, FAS, FLNA, HSC70, OGT (positive control), and actin (loading/negative control) were detected by Western blotting. B, anti-FAS antibody (IP: FAS; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous FAS from NETN cell lysates (250 μg), of which 3% (input) and 60% (immunoprecipitate) were analyzed by SDS-PAGE. FAS, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. C, U2OS cells were transfected with pcDNA3.1 (control) or pCMV-SPORT6 V5-FAS (test). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (300 μg), of which 1.7% (input) and 33.3% (immunoprecipitate) were analyzed by SDS-PAGE. V5, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. A–C, FAS (CST) and FAS (NB) represent anti-FAS antibody from Cell Signaling Technology and Novus Biologicals, respectively. To ensure that images were in the linear range, Western blot exposures from the input and immunoprecipitated fractions are often different. The exposure lengths for the test and control isotype antibody immunoprecipitates are always identical. The migration of molecular mass (MW) markers is indicated.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Control, SDS Page, Positive Control, Negative Control, Western Blot, Transfection, Immunoprecipitation, Migration

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA exhibits reduced catalytic activity when bound to FAS. U2OS cells stably overexpressing pcDNA3.1 (control) or pcDNA3.1 V5-FAS (test) were treated with vehicle (V) or H2O2 (2.5 mm, 2 h). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (1.6 mg). n = 3. A and B, V5, OGA, and actin (loading/negative control) were detected by Western blotting. Western blots for each antibody were exposed for equal lengths of time. A, analysis of the inputs and unbound fractions. B, analysis of the bound fractions (31.25%). C, OGA activity was measured in the inputs and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min and then normalized to the pcDNA3.1 vehicle-treated sample. D, OGA activity was assessed in the input, bound (on-bead, 31.25%), and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min/μg using densitometric analysis of OGA in the input serial dilutions and the bound fractions, and then normalized to the pcDNA3.1 vehicle-treated sample. Only the data from FAS-overexpressing cells treated with H2O2 are shown, as OGA protein and activity was absent in the bound fraction of the other samples. Data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Tukey's MCT, and differences were considered statistically significant at p ≤ 0.0001 (****). The migration of molecular mass (MW) markers is indicated.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Activity Assay, Stable Transfection, Control, Negative Control, Western Blot, Fluorescence, Migration

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: FAS overexpression increases O-GlcNAcylation during oxidative stress. U2OS cells stably overexpressing pcDNA3.1 or pcDNA3.1 V5-FAS were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 5. A, NETN lysates (∼7.5 μg) were analyzed by SDS-PAGE. O-GlcNAc, OGA, OGT, V5, and actin were detected by Western blotting. Total protein stain (colloidal Coomassie G-250) was used to assess protein load. The migration of molecular mass (MW) markers is indicated. B, quantitation of O-GlcNAc levels normalized to G-250. C, quantitation of OGA expression normalized to G-250. D, quantitation of OGT expression normalized to G-250. B–D, data are presented as the mean ± S.E. Significance was determined by RM-2ANOVA followed by Sidak's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Over Expression, Stable Transfection, SDS Page, Western Blot, Staining, Migration, Quantitation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Proposed model for the regulation of OGA during oxidative stress resulting in elevated levels of O-GlcNAc. O-GlcNAc levels become elevated during oxidative stress, and this is associated with cytoprotection. Counterintuitively, in U2OS cells exposed to oxidative stress, OGT activity remains constant, whereas OGA activity and expression are elevated. Our data support a model in which OGA forms complexes with other proteins (?), such as FAS, that regulate its activity leading to a stress-induced elevation of O-GlcNAc.

Article Snippet: Cell culture U2OS human osteosarcoma cells and HEPG2 human hepatocellular carcinoma cells (ATCC) were cultured in high glucose (4.5 g/liter) DMEM (Corning, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Corning) in a humidified water-jacketed CO 2 (5%) incubator at 37 °C.

Techniques: Activity Assay, Expressing