Journal: Molecular carcinogenesis

Article Title: Wogonin Inhibits Ovarian Cancer by Activating the AMPK-TET2-5hmC Axis.

doi: 10.1002/mc.23856

Figure Lengend Snippet: FIGURE 6 | The effect of wogonin on AMPK activation, 5hmC, and Tet2 levels in ovarian cancer cells. Western blot analysis was performed to determine the amount of phosphorylated AMPK (p‐AMPK) and total AMPK in (A) A2780 and (B) Kuramochi ovarian cancer cell lines treated with various concentrations of wogonin. β‐actin served as a loading control. Quantitative analysis of phosphorylated AMPK (relative to total AMPK) is shown in the right panel. Dot blot analysis was performed to determine the effect of wogonin on the amount of 5hmC in (C) A2780 and (D) Kuramochi ovarian cancer cells. Methylene blue staining was used to determine the amount of DNA on the membrane (lower panel). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Primary antibodies against AMPKα (Cell Signaling, Danvers, MA), Phospho‐AMPKα (Thr172, Cell Signaling), Tet2 (Novus Biologicals, Centennial, CO), and β‐Actin (Sigma‐Aldrich) were diluted with 5% nonfat milk in 1× PBST and added to the membrane followed by incubation overnight FIGURE 1 | The effect of wogonin on the proliferation and migration of ovarian cancer cells. (A) A2780 and (B) Kuramochi ovarian cancer cells were treated with various concentrations of wogonin for 24–72 h. Using the Cell Counting Kit‐8 (CCK8), the effect of different concentrations of wogonin on the proliferation of these two ovarian cancer cell lines was investigated.

Techniques: Activation Assay, Western Blot, Control, Dot Blot, Staining, Membrane

Journal: Molecular carcinogenesis

Article Title: Wogonin Inhibits Ovarian Cancer by Activating the AMPK-TET2-5hmC Axis.

doi: 10.1002/mc.23856

Figure Lengend Snippet: FIGURE 7 | The effect of wogonin on Tet2 stability in ovarian cancer cells. (A) A2780 and (B) Kuramochi ovarian cancer cells were first treated with cycloheximide (CHX) to inhibit translation. TET2 expression in cells treated with or without wogonin was then determined using western blot analysis. Quantitative analysis of TET2 level (normalized with actin) was shown in the lower panel. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Primary antibodies against AMPKα (Cell Signaling, Danvers, MA), Phospho‐AMPKα (Thr172, Cell Signaling), Tet2 (Novus Biologicals, Centennial, CO), and β‐Actin (Sigma‐Aldrich) were diluted with 5% nonfat milk in 1× PBST and added to the membrane followed by incubation overnight FIGURE 1 | The effect of wogonin on the proliferation and migration of ovarian cancer cells. (A) A2780 and (B) Kuramochi ovarian cancer cells were treated with various concentrations of wogonin for 24–72 h. Using the Cell Counting Kit‐8 (CCK8), the effect of different concentrations of wogonin on the proliferation of these two ovarian cancer cell lines was investigated.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: 14-3-3 isoforms interact with TET2. A, peptide counts from LC-MS analysis of immunoprecipitates from WT and S99A TET2 (N-terminal 400 amino acids). Analysis of control IgG control is also included. B and C, Myc-TET2 WT and Myc-TET2 S99A were immunoprecipitated from HEK293T cells transiently transfected with the respective plasmids (8 μg of WT TET2, 10 μg of S99A TET2) and analyzed by IB for 14-3-3-θ (B) and 14-3-3-ϵ isoforms (C). Whole-cell extracts were used as inputs. Control IgG and Myc immunoprecipitations were performed from Myc-TET2 and CV-transfected cells, respectively, as negative controls. D, HEK293T cell lysate was immunoprecipitated with anti-TET2 and control IgG antibodies and analyzed by immunoblotting for endogenous 14-3-3 interaction. Data are representative of two independent experiments. E, sequence homology of TET2 around Ser-99 among different species demonstrating a conserved phosphoserine 14-3-3 binding motif ((R/K)XXpSXP). F, WT and S99A TET2 were immunoprecipitated from HEK293T and analyzed by IB with phosphoserine 14-3-3–binding motif antibody.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Immunoprecipitation, Transfection, Western Blot, Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: WT TET2 is more stable than S99A mutant TET2. A, HEK293T cells (one 10-cm plate) were transfected with Myc-TET2 WT (8 μg) or Myc-TET2 S99A (10 μg). After 36 h, cells were incubated with 50 μg/ml CHX. Whole-cell extracts were collected at the indicated time points and analyzed by IB. The figure is a representative of three biological replicates. Ratios of Myc band intensities normalized to actin band intensities are shown at the top. B, normalized Myc band intensities from biological replicates (n = 3) of A plotted over time. One-way repeated-measures ANOVA was used to compare the difference between the two proteins over time (p = 0.0153; F = 16.49). Post hoc analyses were also performed at individual time points using Student's t test: *, p = 0.0226; #, p = 0.0347; **, p = 0.0121. C, HEK293T cells were transfected with WT and S99A TET2. After 24 h, transfected cells were then treated with either vehicle (untreated), 50 μm calpeptin, or 100 nm bortezomib for 24 h followed by immunoblotting of whole-cell extracts. Ratios of Myc band intensities normalized to GAPDH band intensities are shown at the top. D, quantification of data from C (n = 3 biological replicates). Data were analyzed by one-way ANOVA followed by post hoc Tukey's honestly significant difference test. Left, ANOVA p value is 0.1817 (F = 2.2966). Right, ANOVA p value is 0.0198 (F = 8.0966). Post hoc analysis p values are demonstrated. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Mutagenesis, Transfection, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: AMPK phosphorylation at Ser-99 promotes TET2 stability. A, sequence analysis of TET2 at Ser-99 relative to known AMPK substrates. Hyd, hydrophobic. The yellow box denotes phosphorylated residues. B, HEK293T cells (one 10-cm plate) were co-transfected with Myc-TET2 (8 μg) and FLAG-AMPK (10 μg) constructs. Protein extracts were immunoprecipitated using either anti-Myc, anti-FLAG, or control IgG and analyzed by IB. C, in vitro kinase assay utilizing N terminus of recombinant TET2 (rTET2; aa 1–350, ∼75 kDa, GST-tagged) as substrate. Phosphorylation was detected with phosphoserine 14-3-3–binding motif antibody. Results are representative of two independent experiments. D and E, HEK293T cells were transfected with Myc-TET2, followed by treatment with or without metformin for 2 h. Myc-TET2 was immunoprecipitated and analyzed by IB with phosphoserine 14-3-3–binding motif antibody. Data from three biological replicates are quantified in E. F, whole-cell lysates from AMPK WT (+/+) and AMPK KO (−/−) MEFs were collected after treatment with the indicated agent (24-h treatment) and analyzed by IB. DMSO-treated cells were set as control. Ratios of Myc band intensities normalized to actin band intensities are shown at the top. G, global 5hmC levels of genomic DNA extracted from HEK293T cells transfected with WT (8 μg) or S99A mutant TET2 (10 μg) plasmids. Methylene blue (MB) blots are included for loading control. 5hmC quantification from three biological replicates is provided below. I, global 5hmC levels of genomic DNA extracted from AMPK (+/+) MEF cells treated with DMSO (control) or AICAR for 24 h. 5hmC quantification from three biological replicates is provided below. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Phospho-proteomics, Sequencing, Transfection, Construct, Immunoprecipitation, Control, In Vitro, Kinase Assay, Recombinant, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: Disruption of 14-3-3 interaction reduces TET2 phosphorylation and stability. A, endogenous TET2 levels in HEK293T cells transfected with control vector (8 μg) or difopein construct (8 μg). Data from three biological replicates are quantified. B, analysis of Myc TET2 WT and Myc-TET2 S99A by immunoblotting from HEK293T cells transfected with different combinations of Myc-TET2 WT (8 μg), Myc-TET2 S99A (8 μg), difopein (8 μg), and CV (8 μg) constructs as displayed in the figure. The figure is a representative of three biological replicates. C, IP analysis of proteins extracted from HEK293T cells transfected with either WT Myc-TET2 and CV (8 μg of TET2 and 8 μg of CV) or with WT Myc-TET2 and difopein (12 μg of TET2 and 8 μg of difopein). S and L, short and long exposures, respectively, following immunoblotting with phosphoserine 14-3-3–binding motif antibody. D, HEK293T cells were transfected with either CV or difopein construct. After 24 h, cells were treated with CHX (50 μg/ml) and harvested at the indicated time points, followed by analysis of endogenous TET2. E, HEK293T cells (one 10-cm plate) were co-transfected with either WT Myc-TET2 (8 μg) and CV (10 μg) (left) or Myc-TET2 WT (10 μg) and difopein (10 μg) constructs (right). After 24 h, cells were treated with CHX (50 μg/ml) and harvested at the indicated time point, followed by IB using anti-Myc antibody. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Disruption, Phospho-proteomics, Transfection, Control, Plasmid Preparation, Construct, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: 14-3-3 protects Ser-99 phosphorylation of TET2 in vitro. A, schematic representation of the workflow for phosphatase protection assay. B, representative Western blot analysis of Myc-TET2 immunoprecipitates following in vitro R18 treatment. C, densitometry ratios of phosphorylated TET2/total Myc-TET2 (left) and total 14-3-3/total Myc-TET2 (right) were calculated from three biological replicates and plotted. Data were analyzed by one-way ANOVA followed by post hoc Tukey's honestly significant difference test. Left, ANOVA p value is 0.0146 (F = 9.2774). Post hoc analysis p values are demonstrated. Right, ANOVA p value is 0.0044 (F = 15.2941). Post hoc analysis p values are demonstrated. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Phospho-proteomics, In Vitro, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: PP2A dephosphorylates TET2 at Ser-99. A, HEK293T cells were transfected with Myc-TET2 followed by IP with anti-Myc, anti-TET2, or IgG control antibodies. IPs were analyzed by IB for the indicated proteins. B, HEK293T cell lysate was immunoprecipitated by either anti-TET2 or control IgG antibodies and analyzed by immunoblot. C and D, control knockdown and si-RNA (si-1, si-2) knockdown of PP2A-Bα cell lysates were immunoprecipitated with anti-TET2 antibody and analyzed by immunoblotting in C. Whole-cell extracts were used as inputs (D). Levels of TET2 Ser-99 phosphorylation were calculated by measuring the ratio of band intensities of phospho-Ser-99-TET2 (IB: p-14-3-3 motif) and total TET2 (IB: TET2). TET2 band intensities in the whole-cell lysates were normalized to loading control tubulin. E, endogenous TET2 levels in HEK293T cells treated with siPP2A-Bα (construct #1). Data from three biological replicates are quantified. F, immunoprecipitated Myc-TET2 was treated in vitro with or without rPP2A. Ser-99 phosphorylation was assessed by immunoblotting with phosphoserine 14-3-3–binding motif antibody. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Knockdown, Phospho-proteomics, Construct, In Vitro, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: 14-3-3 protects TET2 Ser-99 phosphorylation from PP2A. A and B, HEK293T cells were transfected with Myc-TET2. 24 h following transfection, cells were treated with or without PP2A inhibitor OKA (2-h treatment). Immunoprecipitates and inputs were then analyzed by immunoblotting with the indicated antibodies in A. Levels of Myc-TET2 phosphorylation from three biological replicates are quantified in B. C, HEK293T cells were cotransfected with Myc-TET2 and difopein constructs. Forty-eight hours after transfection, cells were treated with the indicated concentrations of OKA for 2 h. Anti-Myc immunoprecipitates were assessed for Ser-99 phosphorylation with phosphoserine 14-3-3–binding motif antibody. This figure is a representative of two biological replicates. D, HEK293T cells (one 10-cm plate) were cotransfected with either Myc-TET2 (8 μg) and CV (10 μg) (lanes 4–6) or Myc-TET2 (8 μg) and difopein (10 μg) constructs (lanes 1–3). After 48 h, cells were treated with either AICAR (1 mm) or metformin (5 mm) for 2 h before harvesting and analysis of Myc IPs and inputs. E, HEK293T cells were cotransfected with Myc-TET2 and difopein. After 48 h, cells were treated with the indicated treatment for 2 h, followed by analysis of Myc IPs and inputs. F and G, cell lysates from HEK293T cells treated with the indicated agents for 48 h were analyzed by immunoblotting. Total TET2 levels were normalized to loading control actin, and ratios are indicated at the top. Data from three biological replicates are quantified in G. Data were analyzed by one-way ANOVA followed by post-hoc Tukey's honestly significant difference test. ANOVA p value is 0.0101 (F = 10.9000). Post hoc analysis p values are demonstrated. Error bars, S.E.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques: Phospho-proteomics, Transfection, Western Blot, Construct, Binding Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation

doi: 10.1074/jbc.RA119.011089

Figure Lengend Snippet: Proposed model for dynamic regulation of TET2 by 14-3-3.

Article Snippet: These antibodies include 1) c-Myc (#M4439; origin, mouse; source, monoclonal; 1:3000), FLAG (#F1804; origin, mouse; source, monoclonal; 1:3000), PP2A-Bα (#SAB4200241; origin, mouse; source, monoclonal; 1:1000), and tubulin (#T9026; origin, mouse; source, monoclonal; 1:5000) from Sigma-Aldrich; 2) 14-3-3-θ from Proteintech (#14503–1-AP; origin, rabbit; source, polyclonal; 1:1000); 3) β-actin conjugated with HRP (#ab49900; origin, mouse; source, monoclonal; 1:10,000) and GAPDH (#ab181602; origin, rabbit; source, polyclonal; 1:5000) from Abcam; 4) anti-5hmC from Active Motif (#39069; origin, rabbit; source, polyclonal; 1:2500); 5) anti-TET2 from Diagenode (#C15200179; origin, mouse; source, monoclonal; 1 μg/1 mg lysate for immunoprecipitation); and 6) 14-3-3 isoform sampler kit (#9769; origin, rabbit; source, monoclonal and polyclonal; 1:1000), phosphoserine 14-3-3–binding motif (#9601; origin, rabbit; source, monoclonal; 1:1000), AMPKα (#5831; origin, rabbit; source, monoclonal; 1:1000), phospho-AMPKα (#2535; origin, rabbit; source, monoclonal; 1:1000), phospho-ACC (#3661; origin, rabbit; source, monoclonal; 1:1000), anti-protein phosphatase 2A (#9780; origin, rabbit; source, monoclonal; 1:1000) sampler kit, and TET2 (#18950; origin, rabbit; source, monoclonal; 1:1000) from Cell Signaling Technology.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effect of oxLDL on TET2 expression and the CSE/H 2 S system in HUVECs. (A–D) OxLDL reduced TET2 mRNA and protein expression in HUVECs at the concentration-dependent fashion (0, 25, 50, 75, and 100 μg/ml). 75 μg/ml oxLDL decreased TET2 mRNA and protein expression in HUVECs at the time-dependent manner (0, 12, 24, and 48 h). ∗ P < 0.05, ∗∗ P < 0.01 vs. 0 μg/ml oxLDL group (A,B) or vs. 0 h group (C,D) . (E–H) OxLDL reduced CSE mRNA and protein expression in HUVECs at the concentration-dependent fashion (0, 25, 50, 75, and 100 μg/ml) and 75 μg/ml oxLDL decreased CSE mRNA and protein expression in HUVECs at the time-dependent fashion (0, 12, 24, and 48 h). ∗ P < 0.05, ∗∗ P < 0.01 vs. 0 μg/ml oxLDL group (E,F) or vs. 0 h group (G,H) . (I,J) The effects of increasing concentrations of oxLDL on the H 2 S production rates in cells and the effects of 75 μg/ml oxLDL on H 2 S production rates in cells over time. ∗ P < 0.05, ∗∗ P < 0.01 vs. 0 μg/ml oxLDL group (I) or vs. 0 h group (J) . (K) Detection of intracellular H 2 S levels in HUVECs with or without 75 μg/ml oxLDL treatment for 24 h using H 2 S-specific fluorescent probes. Representative fluorescent images were taken using a fluorescent microscope. Scale bar = 20 μm. All results are expressed as the mean ± SD of three independent experiments.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Expressing, Concentration Assay, Microscopy

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effects of TET2 on the oxLDL-induced dysfunction of HUVECs. (A) The effects of TET2 overexpression and shRNA plasmids on TET2 protein expression in HUVECs. (B,C) HUVECs were transduced with TET2 overexpression plasmid or TET2 shRNA plasmid, then treated with 75 μg/ml oxLDL for 24 h. THP-1 cells were seeded onto HUVECs and co-cultured for 30 min. After washing the non-adherent cells, adherent cells were detected and counted under a light microscope. Representative light microscopic pictures of THP-1 cell adhesion to oxLDL-activated HUVECs (B) and quantitative analysis of adhesion results (C) . Data are the mean ± SEM of results from at least three independent experiments, each performed in duplicate. ∗ P < 0.05, ∗∗ P < 0.01 vs. control (treated with oxLDL alone) group. (D–G) The mRNA and protein levels of ICAM-1 and VCAM-1 in oxLDL-treated HUVECs with TET2 overexpression or TET2 silencing were determined by real-time PCR and western blot analyses. All results are expressed as the mean ± SD of three independent experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. control (treated with oxLDL alone) group. All results are expressed as the mean ± SD of three independent experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. control group.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Over Expression, shRNA, Expressing, Transduction, Plasmid Preparation, Cell Culture, Light Microscopy, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effects of TET2 on the CSE/H 2 S system in oxLDL-induced dysfunction of HUVECs. HUVECs were transfected with or without TET2 overexpression plasmid or TET2 shRNA plasmid in the presence of oxLDL for 24 h. The expression of CSE mRNA (A) and protein (B) was examined via real-time PCR and western blot analyses in cells. (C) The H 2 S production rates in each group of cells were determined as described in “Materials and Methods” section. (D) Representative fluorescent images of intracellular H 2 S levels in each group of cells using H 2 S-specific fluorescent probes. Scale bar = 20 μm. All results are expressed as the mean ± SD of three independent experiment. ∗ P < 0.05, ∗∗ P < 0.01 vs. control group.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, shRNA, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effects of TET2 on NF-κB activation in oxLDL-induced dysfunction of HUVECs. HUVECs were transfected with or without TET2 overexpression plasmid or TET2 shRNA plasmid in the presence of oxLDL for 24 h. The levels of IkBα protein (A) and nuclear NF-κB p65 protein (B) , respectively, were evaluated by western blot analysis in each group of cells. All results are expressed as the mean ± SD of three independent experiment. ∗ P < 0.05, ∗∗ P < 0.01 vs. control group. (C) Distribution of NF-κB p65 protein expression was detected by immunostaining in each group of cells. NF-κB p65 positive staining is red. NF-κB p65 accumulation in the nuclei of the cells shows pink. DAPI staining is blue. Scale bar = 20 μm.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, shRNA, Western Blot, Control, Expressing, Immunostaining, Staining

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effects of the CSE/H 2 S system on the TET2-induced improvement of oxLDL-treated dysfunction of HUVECs. HUVECs were transfected with or without TET2 overexpression plasmid or TET2 overexpression plasmid + CSE siRNA in the presence of oxLDL for 24 h. (A,B) Representative microscopic images of the adhesion of THP-1 cells to HUVECs (A) and quantitative analysis of adhesion results. Data are the mean ± SEM of results from at least three independent experiments. (B). The ICAM-1 (C) and VCAM-1 (D) proteins were evaluated by western blot analysis in each group of cells. The levels of IkBα protein (E) and nuclear NF-κB p65 protein (F) , respectively, were examined by western blot analysis in each group of cells. (G) Distribution of NF-κB p65 protein expression was detected by immunostaining in each group of cells. NF-κB p65 positive staining is red. NF-κB p65 accumulation in the nuclei of the cells shows pink. DAPI staining is blue. Scale bar = 20 μm. All results are expressed as the mean ± SD of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 vs. oxLDL-treated alone group. ## P < 0.01 vs. TET2 overexpression plasmid-treated group.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Expressing, Immunostaining, Staining

Journal: Frontiers in Pharmacology

Article Title: TET2 Protects against oxLDL-Induced HUVEC Dysfunction by Upregulating the CSE/H 2 S System

doi: 10.3389/fphar.2017.00486

Figure Lengend Snippet: Effects of TET2 on the methylation level of the CSE promoter in oxLDL-treated HUVECs. (A) The features of CpG Island in human CSE gene promoter region was analyzed using the UCSC Human Genome Browser ( http://genome.ucsc.edu/ ). (B) A schematic diagram of the CpG dinucleotides within the CSE promoter. The nucleotide number is relative to the transcription start site of CSE. The red line indicates the region that was tested with BSP. (C) HUVECs were transfected with or without TET2 overexpression plasmid or TET2 shRNA plasmid in the presence of oxLDL for 24 h. The methylation levels of the CSE promoters in each group of cells were determined by BSP method. Each row represents an individual clone sequenced; black and white circles represent methylated and unmethylated CpGs sites, respectively. The number of methylated CpGs divided by the whole CpG sites examined is given as a percentage of methylation. (D) Immunostaining for 5hmC in each group of cells. 5hmC is red, DAPI staining is blue, n = 4. Scale bar = 20 μm.

Article Snippet: The primary antibodies used include GAPDH (1:1000, Hangzhou Goodhere Biotechnology Co., Ltd.), TET2 (1:1000, Proteintech), CSE (1:1000, Proteintech), ICAM-1 (1:1000, Proteintech), VCAM-1 (1:500, Santa Cruz), IκBα (1:1000, Proteintech), NF-κB p65 (1:1000, Proteintech), and Histone H3 (1:5000, Abcam).

Techniques: Methylation, Transfection, Over Expression, Plasmid Preparation, shRNA, Immunostaining, Staining

Journal: Science advances

Article Title: TET2 coactivates gene expression through demethylation of enhancers.

doi: 10.1126/sciadv.aau6986

Figure Lengend Snippet: Fig. 1. TET2 is a coactivator of ER. (A) TET2 and ER protein levels were determined by Western blotting in TET2-WT and TET2-KO CAL51 cells (ER−) and MCF7 cells (ER+). HSP90 was used as an internal control (n = 3). (B) Cells (2 × 104) of each cell line were seeded in six-well plates, and the cell growth ability of TET2-WT and TET2-KO cells was determined by cell counting at the indicated days. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Representative images of the cell morphology of MCF7-TET2-WT and MCF7-TET2-KO clones (n = 3). (D) MCF7-TET2-WT and MCF7-TET2-KO cells were maintained in phenol red–free medium containing 5% charcoal-stripped fetal bovine serum (FBS) for 4 days. Subsequently, 5 × 104 cells were seeded in six-well plates in the presence of dimethyl sulfoxide (DMSO) or E2 at 10 nM, and cell growth was determined by cell counting at the indicated days. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (E) Schematic of the RNA-seq experimental workflow using MCF7-TET2-WT or MCF7-TET2-KO cells treated with either DMSO or E2 (10 nM) for 4 hours (n = 2). (F and G) Venn diagrams (F) and heat maps (G) showing the overlap of genes induced by E2 between TET2-WT and TET2-KO cells. Log2FC, log2 fold change. (H) Representative RNA-seq tracks of genes differentially induced by E2 in TET2-WT and TET2-KO cells (n = 2).

Article Snippet: ER (sc-543) and HSP90 (sc-7947) were purchased from Santa Cruz Biotechnology, and TET2 [A304-247A, for Western blotting and immunoprecipitation (IP)] was purchased from Bethyl Laboratories.

Techniques: Western Blot, Control, Cell Counting, Two Tailed Test, Clone Assay, RNA Sequencing

Journal: Science advances

Article Title: TET2 coactivates gene expression through demethylation of enhancers.

doi: 10.1126/sciadv.aau6986

Figure Lengend Snippet: Fig. 2. Enhancer TET2 mediates proper recruitment of ER. (A) TET2-WT and TET2-KO MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 at 100 nM for 45 min. Heat maps generated from ChIP-seq data showing the occupancy of ER in DMSO- and E2-treated cells. All rows are centered on ER peaks and further divided into TSS and non-TSS regions. TSS and non-TSS regions were further divided into three clusters each by k-means (n = 2). (B) Left: Log2 fold change heat map shows the comparison of ER occupancy between TET2-WT and TET2-KO cells treated with DMSO versus E2. Right: Log2 (fold change) of nearby gene expression in Tet2-WT or Tet2-KO cells treated with either DMSO or E2 (n = 2). (C) Heat maps generated from ChIP-seq data showing the occupancy of TET2, which is centered on the ER peaks, and rows ordered as in (A) (n = 2). (D) Representative genome browser tracks of TET2 and ER occupancy at enhancers. (E) Venn diagram showing common peaks between total TET2 peaks and ER cluster 1 peaks. (F) Box plot quantifying changes on H3K4me3, H3K4me1, H3K27me3, and H3K27Ac occupancy at ER alone and ER/TET2 co-occupied cluster 1 peaks. (G to J) Well-observed CpG methylation around ER-binding sites overlapping TSS sites (G) and non-TSS regions (H to J) separated by clusters identified in (A). Average CpG methylation values ± SEM for two biological replicates per cell type are plotted for the center of ER-binding sites ± 2500 base pairs (bp). NTD, N-terminal domain.

Article Snippet: ER (sc-543) and HSP90 (sc-7947) were purchased from Santa Cruz Biotechnology, and TET2 [A304-247A, for Western blotting and immunoprecipitation (IP)] was purchased from Bethyl Laboratories.

Techniques: Generated, ChIP-sequencing, Comparison, Gene Expression, CpG Methylation Assay, Binding Assay

Journal: Science advances

Article Title: TET2 coactivates gene expression through demethylation of enhancers.

doi: 10.1126/sciadv.aau6986

Figure Lengend Snippet: Fig. 3. Positive feedback between TET2 and ER at enhancers. (A) Parental MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 at 10 nM for 4 hours. Heat maps generated from RNA-seq data showing the expression changes of selected epigenetic factors including histone and DNA modifiers (n = 2). (B) Real-time PCR was performed to determine the expression of TET1 to TET3 after E2 treatment. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Parental MCF7 cells were maintained in phenol red–free medium contain- ing 5% charcoal-stripped FBS for 4 days, followed by E2 treatment for the indicated times. TET2 protein levels were determined by Western blotting. HSP90 was used as an internal control (n = 3). (D) Parental MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 (100 nM) for 45 min. Representative genome browser tracks showing the occupancy of ER, H3K4me3, H3K27ac, and H3K4me1 levels at TET2 (n = 2). MCF7 cells were treated with tamoxifen (1 M) for 72 hours. (E) Protein levels of TET2 were determined by Western blotting. HSP90 was used as an internal control (n = 3). (F) Representative RNA-seq tracks showing TET2 expression changes in response to tamoxifen treatment (n = 2). (G and H) Representative ChIP-seq tracks (G) and average plot (H) showing loss of TET2 chromatin occupancy in response to tamoxifen treatment.

Article Snippet: ER (sc-543) and HSP90 (sc-7947) were purchased from Santa Cruz Biotechnology, and TET2 [A304-247A, for Western blotting and immunoprecipitation (IP)] was purchased from Bethyl Laboratories.

Techniques: Generated, RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Control, ChIP-sequencing

Journal: Science advances

Article Title: TET2 coactivates gene expression through demethylation of enhancers.

doi: 10.1126/sciadv.aau6986

Figure Lengend Snippet: Fig. 4. Loss of MLL3, but not MLL4, COMPASS disrupts the TET2-ER axis. (A) Cartoon of MLL3 and MLL4 COMPASS regulation of gene expression from enhancers. (B) Level of expression of MLL3, MLL4, and TET2, as assessed by real-time PCR in MCF7 cells treated with shNONT, shMLL3, and shMLL4. Data are means ± SD; n = 3 inde- pendent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Protein levels of MLL3, MLL4, and TET2 as determined by Western blotting in MCF7 cells treated with shNONT, shMLL3, and shMLL4 (n = 3). (D) Heat maps of MLL3, H3K4me1, and H3K27ac log2 fold changes in response to E2 treatment. Rows are centered on the non- TSS ER peaks and ordered as in Fig. 2A (n = 2). (E) Representative genome browser tracks showing the occupancy of MLL3 at TET2 enhancers (n = 2). (F) Representative tracks showing the recruitment of MLL3 and increased occupancy of H3K4me1 at TET2 enhancers induced by E2 treatment (n = 2). (G) Representative RNA-seq tracks showing the expression of TET2 induced by E2 in shNONT and shMLL3 cells (n = 2).

Article Snippet: ER (sc-543) and HSP90 (sc-7947) were purchased from Santa Cruz Biotechnology, and TET2 [A304-247A, for Western blotting and immunoprecipitation (IP)] was purchased from Bethyl Laboratories.

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, RNA Sequencing

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 1: Distribution of TET2, 5hmC, and 5mC in atherosclerotic lesions and normal vascular tissue. Immunostaining was performed using antibodies against TET2, 5hmC, and 5mC in normal vascular and atherosclerotic tissues in ApoE−/− mice (n = 10 each). Cell nuclei were counterstained with DAPI. Scale bars are 200 and 50 μm.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Immunostaining

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 2: TET2 inhibited the atherosclerotic lesion progression of high fat-fed ApoE−/− mice. A. Representative en face preparations of the total aorta in ApoE−/− mice treated with TET2 overexpression or TET2 shRNA lentiviruses fed a high-fat diet for 12 weeks. B.-C. Representative microscopic images and quantification of atherosclerotic plaque areas in cross-sections of aortic roots with Oil-red staining in ApoE−/− mice treated with TET2 overexpression or TET2 shRNA lentiviruses fed a high-fat diet for 12 weeks. Values are mean ± SEM (n = 20 each). D.-E. Representative microscopic images of the aortic sinus with Masson’s Trichrome staining and immunostaining for CD68 (a macrophage marker) in ApoE−/− mice treated with TET2 overexpression or TET2 shRNA lentiviruses fed a high-fat diet for 12 weeks. **P < 0.01 compared with the control group. TET2 OE:TET2 overexpression.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Over Expression, shRNA, Staining, Immunostaining, Marker, Control

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 3: TET2 inhibited inflammation factor expression in the atherosclerotic lesions of high fat-fed ApoE−/− mice. Representative micrographs of immunofluorescence staining of ICAM-1, VCAM-1, IL-1β, and MCP-1 in atherosclerotic plaques were derived from the control, TET2 OE, and TET2 shRNA ApoE−/− mice fed with a high-fat diet for 12 weeks (n = 10 each). Scale bar is 50 μM. TET2 OE:TET2 overexpression.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Expressing, Immunofluorescence, Staining, Derivative Assay, Control, shRNA, Over Expression

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 4: TET2 improved autophagy in the atherosclerotic lesions of high-fat-fed ApoE−/− mice. A. Representative micrographs of immunofluorescence staining of autophagy markers, such as Beclin 1, LC3, and p62, in atherosclerotic plaques were derived from the control, TET2 OE, and TET2 shRNA ApoE−/− mice fed with high-fat diets for 12 weeks. DAPI was used for nuclear stains (blue). Scale bar is 50 μM. B. Representative Western blots showing the levels of Beclin 1, LC3, and p62 in aortic arch in the different groups. C.-E. Quantitative data showing the levels of autophagy-related proteins in aortic arch in the different groups. All results are expressed as mean ± S.D. (n = 3 each). *P < 0.05, **P < 0.01 versus control group. TET2 OE:TET2 overexpression.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Immunofluorescence, Staining, Derivative Assay, Control, shRNA, Western Blot, Over Expression

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 5: TET2 increased 5hmC level and decreased 5mC level in ox-LDL-treated HUVECs. A. Representative immunostaining micrographs show that TET2 OE enhanced 5hmC level and reduced 5mC level in HUVECs treated with 50 mg/L ox- LDL for 24 h. Consistently, treatment with TET2 shRNA reduced 5hmC generation and increased 5mC generation. DAPI was used for nuclear staining (blue). Scale bar is 20 μM. B.-C. TET2 levels were determined in HUVECs with different treatments using Western blot. Values are mean ± SD from three independent experiments, each performed in triplicate. **P < 0.01 versus control group. TET2 OE:TET2 overexpression.TET2 OE:TET2 overexpression.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Immunostaining, shRNA, Staining, Western Blot, Control, Over Expression

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 6: TET2 improved autophagy and autophagic flux in ox-LDL-treated HUVECs. A. Representative Western blots showing the levels of Beclin 1, LC3 and p62 in HUVECs with different treatments. B. Quantitative data showing the levels of autophagy- related proteins in HUVECs with different treatments. All results are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01 versus ox-LDL-treated group. C. Representative images showing LC3 localization in HUVECs from different groups infected with the GFP-RFP-LC3 adenovirus for 24 h. Scale bar is 5 μM. D. Bisulfite sequence analysis was performed to compare the DNA methylation at the Beclin 1 promoter.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Western Blot, Infection, Sequencing, DNA Methylation Assay

Journal: Oncotarget

Article Title: Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE-/- mice.

doi: 10.18632/oncotarget.13121

Figure Lengend Snippet: Figure 7: TET2 inhibited the expression of inflammatory factors in ox-LDL-treated HUVECs. The increased levels of VCAM-1, ICAM-1, IL-1β, and MCP-1 in response to ox-LDL treatment were reduced by TET2 OE and further elevated by TET2 shRNA. Scale bar is 20 μM.

Article Snippet: TET2 overexpression or knockdown in endothelial cells HUVECs were seeded at 4×105 cells/well in a six-well plate and then transfected with 5 μg of TET2 overexpression plasmid or TET2 shRNA plasmid (OriGene Technologies Inc.) using Lipofectamine LTX (Invitrogen) in accordance with the manufacturer’s instruction.

Techniques: Expressing, shRNA

Journal: Journal of Advanced Research

Article Title: Gut microbial GABAergic signaling improves stress-associated innate immunity to respiratory viral infection

doi: 10.1016/j.jare.2023.06.008

Figure Lengend Snippet: GABA acts through the αKG/Tet2/ PPARγ axis to mediate metabolic-epigenetic regulation of AMs. (A) Schematic diagram of the TCA cycle, with the key enzymes regulated upon stress labeled. Red arrow, down-regulated, and Green arrow, up-regulated in stressed AMs versus non-stressed AMs post IAV infection. (B) Serum levels of αKG and succinate analyzed by GC–MS, with the ratio of αKG to succinate provided. (C-F) Stressed and Non-stressed (NS) mice were infected or uninfected with IAV for 3 days. CD45 + CD11b - CD64 + CD11c + SiglecF + AMs were sorted for subsequent analysis. (C) Immunoblotting of Tet2; (D) Dot-blot analysis of 5hmc or 5mc level. MB, methylene blue staining (as loading control). (E) ChIP test of 5hmc or 5mc enrichment at the pparγ locus; (F) Immunoblotting of PPARγ; (G) Immunoblotting of Tet2 and PPARγ in MH-S cells treated with GABA at the indicated doses; (H) Immunoblotting of Tet2 or PPARγ in GABA-treated macrophages with or without bicuculline (Bic; 40 μM). (I-K) MH-S cells were transfected with Tet2 siRNA (siTet2) or siRNA (siNC), and then treated with GABA (100 μM) or 0.01% DMSO prior to IAV infection. (I) PPARγ luciferase activity; (J) Immunoblotting of Tet2 and PPARγ; (K) Heatmapping of AM-associated genes. Shown are representative images. The bar graph is the densitometric quantification of western blot bands. Data from three individual experiments are shown as means ± SD. * P < 0.05, ** P < 0.01, with one-way ANOVA (three or more groups) followed by Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies against Tet2 (#92529), PPARγ (#2443), and β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Labeling, Infection, Gas Chromatography-Mass Spectrometry, Western Blot, Dot Blot, Staining, Control, Transfection, Luciferase, Activity Assay