Journal: Oncotarget

Article Title: Glycolytic metabolism influences global chromatin structure

doi:

Figure Lengend Snippet: (A) HDAC inhibitor panobinostat (80 nM) was added to cells treated with or without 2-DG. Histone acetylation was detected by immunoblot with a lysine acetylation specific antibody. HSP90 and β-actin blots serve as loading controls. (B) Transmission electron microscopy analysis was performed in A549 cells treated with control, 2-DG (10 mM), panobinostat (80 nM) or in combination. Scale bar is 5 μm. (C) Immunofluorescent staining with H3K27Ac, or heterochromatin marker HP1α and H3K9me3 antibody in A549 cells treated with 2-DG or transfected with siRNA targeting HK1 , PKM in the presence of panobinostat (80 nM). Scale bar is 5 μm.

Article Snippet: H3K9me3 antibody (Millipore, #07–442); HP1α antibody (cell signaling, #2616); Acetylated-Lysine (Ac-K2–100) antibody (Cell Signaling, #9814); Histone H3 antibody (Cell Signaling, #4499); Histone H4 antibody (Cell Signaling, #2935); H2AK5 antibody (Cell Signaling, #2576); H2BK5 antibody (Cell Signaling, #12799); H3K9Ac antibody (Cell Signaling, #9649); H3K14Ac antibody (Cell Signaling, #7627); H3K27Ac antibody (Cell Signaling, #8173); H3K56Ac antibody (Cell Signaling, #4243); H4K5Ac antibody (Cell Signaling, #8647); H4K8Ac antibody (Cell Signaling, #2594); H4K12Ac antibody (Cell Signaling, #2591); Anti-β-actin antibody (AC-15, Sigma); Anti-Hsp90 antibody (AC-16, Sigma) was used for western blot; 2-Deoxy-D-glucose (2-DG), bleomycin was brought from Sigma; HDAC inhibitor Panobinostat was purchased from LC Laboratories.

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Staining, Marker, Transfection

Journal: Oncotarget

Article Title: Glycolytic metabolism influences global chromatin structure

doi:

Figure Lengend Snippet: (A) Comet assay were performed in A549 cells that were treated with glycolysis inhibitor 2-DG (10 mM, 24 hours), HDAC inhibitor panobinostat (80 nM, 24 hours) or combined as indicated, DNA damage were induced by bleomycin (40 μM) treatment for 20 min at 37°C. Bleomycin was then washed away and the cells were cultured in medium with the indicated chemicals for 4 hours. DNA damage levels were shown as % of DNA in comet tail. Error bars represent mean ± s.e.m. (B) Glycolysis inhibitor 2-DG sensitize A549 cells to DNA damage drugs. A549 cells were treated with control or 2-DG, bleomycin or combination for 24 hours. Quantified cell viabilities of three independent experiments are shown as mean ± s.d. (C) Proposed model for glycolysis regulated global chromatin structure changes. Glycolysis stimulates global histone acetylation by repressing HDAC through increased production of pyruvate, lactate; and by stimulating HAT activity via increased cellular concentration of Acetyl-CoA. Increased global histone acetylation stimulates the open chromatin structure, which contributes to the efficient DNA repair, and chemoresistance.

Article Snippet: H3K9me3 antibody (Millipore, #07–442); HP1α antibody (cell signaling, #2616); Acetylated-Lysine (Ac-K2–100) antibody (Cell Signaling, #9814); Histone H3 antibody (Cell Signaling, #4499); Histone H4 antibody (Cell Signaling, #2935); H2AK5 antibody (Cell Signaling, #2576); H2BK5 antibody (Cell Signaling, #12799); H3K9Ac antibody (Cell Signaling, #9649); H3K14Ac antibody (Cell Signaling, #7627); H3K27Ac antibody (Cell Signaling, #8173); H3K56Ac antibody (Cell Signaling, #4243); H4K5Ac antibody (Cell Signaling, #8647); H4K8Ac antibody (Cell Signaling, #2594); H4K12Ac antibody (Cell Signaling, #2591); Anti-β-actin antibody (AC-15, Sigma); Anti-Hsp90 antibody (AC-16, Sigma) was used for western blot; 2-Deoxy-D-glucose (2-DG), bleomycin was brought from Sigma; HDAC inhibitor Panobinostat was purchased from LC Laboratories.

Techniques: Single Cell Gel Electrophoresis, Cell Culture, Activity Assay, Concentration Assay

Journal: Oncology Letters

Article Title: Epigenetic treatment-mediated modulation of PD-L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD-L1 reverse signaling

doi: 10.3892/ol.2018.9841

Figure Lengend Snippet: In vitro effects on cell growth, mRNA expression levels of PD-1 and PD-L1 of Aza, LBH-589, MCT-3 or a combination of Aza and LBH589 in KG-1 cells. (A) In vitro effects on cell growth in KG-1 cells untreated (Con) or treated for 24 h with Aza, LBH-589 (LBH), MCT-3 or Aza+LBH as assessed by trypan blue staining. n=3. In vitro mRNA expression of (B) PD-1 and (C) PD-L1 mRNA in KG-1 cells untreated (Con) and treated for 24-h treatment with Aza, MCT-3, LBH or a combination of Aza+LBH. *P<0.05 vs. con, **P<0.01 vs. con, n=3-4. PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; Aza, azacytidine; Con, control.

Article Snippet: Agents: Azacytidine (Aza) (Sigma, Australia) and the histone deacetylase inhibitors (HDACi) Panobinostat (LBH-589) (LBH) (Sapphire Bioscience Pty Ltd., Sydney, Australia) and MCT-3 ( 12 ) were added to plates for 24 h. Aza was dissolved in H 2 O with 0.2% acetic acid and used at a final concentration of 1.0 μM.

Techniques: In Vitro, Expressing, Staining, Control

Journal: Oncology Letters

Article Title: Epigenetic treatment-mediated modulation of PD-L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD-L1 reverse signaling

doi: 10.3892/ol.2018.9841

Figure Lengend Snippet: In vitro and in vivo mRNA expression levels of NF-κB and Bcl-xL in KG-1 cells treated with Aza, LBH-589, MCT-3 or a combination of Aza+LBH-589 and from PBMC's from a non-responder and responder patient. (A) NF-κB and (B) Bcl-xL mRNA expression in KG-1 cells untreated (Con) or treated for 24 h with Aza, LBH-589 (LBH), MCT-3 or Aza+LBH *P<0.05 Con vs. Aza and Aza+LBH (n=3) and (C) Bcl-xL mRNA expression in PBMC's from non-responsive patient 010 at day 0, 6 and day 20 (n=1). (D) Bcl-xL and (E) NF-κB mRNA expression in PBMC's from an EGT-responsive patient 007 at day 0, 7 and day 21 (n=1). Responses were defined according to international working group criteria for AML and MDS (15). Aza, azacytidine; PBMC, peripheral blood mononuclear cells; EGT, epigenetic therapy; Con, control; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome.

Article Snippet: Agents: Azacytidine (Aza) (Sigma, Australia) and the histone deacetylase inhibitors (HDACi) Panobinostat (LBH-589) (LBH) (Sapphire Bioscience Pty Ltd., Sydney, Australia) and MCT-3 ( 12 ) were added to plates for 24 h. Aza was dissolved in H 2 O with 0.2% acetic acid and used at a final concentration of 1.0 μM.

Techniques: In Vitro, In Vivo, Expressing, Control

Journal: Analytical Chemistry

Article Title: Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions

doi: 10.1021/acs.analchem.2c00391

Figure Lengend Snippet: I-PISA assay in the lysate allows one to detect direct targets of multitarget drugs. (A) Treatment of the K562 lysate with 10 μM Staurosporine resulting in solubility change of several kinases. (B) I-PISA of the K562 lysate treated with 10 μM Panobinostat revealing known drug targets. (C) Median coefficient of variation (CV) of protein signals between the replicates in all lysate I-PISA assays is below 4%, highlighting the excellent reproducibility of the method. Abbreviations: MTX, methotrexate; Pano, Panobinostat; Stauro, Staurosporine; 4817 proteins were quantified for each panel.

Article Snippet: Next, aiming to test the optimized I-PISA assay for multitarget drugs, we treated the K562 lysate for 15 min with either 10 μM Staurosporine (Stauro) (a pan-kinase inhibitor) or Panobinostat (Pano) (a pan-HDAC inhibitor).

Techniques: Solubility

Journal: Analytical Chemistry

Article Title: Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions

doi: 10.1021/acs.analchem.2c00391

Figure Lengend Snippet: I-PISA assay in intact cells allows one to detect downstream effector proteins. (A) Volcano plot of K562 cells treated with MTX showing additional shifting proteins compared to the lysate experiment (4113 proteins quantified). (B) Comparison of protein solubility alterations between the lysate and cell experiments pinpointing downstream targets in the latter. (C) Treatment of K562 cells with Panobinostat highlighting downstream proteins of direct targets (4113 proteins quantified). (D) Comparison of I-PISA assay Panobinostat results obtained in cells versus lysates providing additional and/or more reliable target information.

Article Snippet: Next, aiming to test the optimized I-PISA assay for multitarget drugs, we treated the K562 lysate for 15 min with either 10 μM Staurosporine (Stauro) (a pan-kinase inhibitor) or Panobinostat (Pano) (a pan-HDAC inhibitor).

Techniques: Comparison, Solubility