Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: Expression of β CR in atherosclerotic lesions of apoE −/− mice. Immunohistochemistry with IgG, anti- β CR, anti-EPOR, or anti-F4/80 was performed and then recognized by the corresponding horseradish peroxidase-conjugated secondaryantibody on aortic specimens of apoE deficient mice. Hematoxylin was used as counterstaining.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: β CR colocalizes with erythropoietin receptor (EPOR) in macrophagesbone-marrow-derived macrophages were treated with vehicle (PBS) or EPO (5 U/mL) for 10 min. Cells were fixed and immunostained with anti- β CR or anti-EPOR Ab for confocal microscopy examination. Nuclei were stained with DAPI.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Confocal Microscopy, Staining

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: EPO treatment does not affect the interaction of β CR and EPOR in macrophages. Macrophages were treated with or without EPO (5 U/mL) for 10 min. Cellular lysates were immunoprecipitated (IP) with (a) anti-EPOR Ab or (b) anti- β CR Ab and then were immunoprobed with anti-EPOR or anti- β CR Ab by immunoblotting (IB). Data are mean ± SEM from 5 independent experiments.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: β CR neutralizing Ab diminishes the EPO-mediated protection of oxLDL-induced lipid accumulation in macrophages. Bone-marrow-derived macrophages were pretreated with IgG (200 ng/mL) or β CR neutralizing Ab (200 ng/mL) for 2 h and then treated with EPO (5 U/mL) for 12 h, followed by oxLDL (50 μ g/mL) for additional 18 h. ((a) and (b)) The intracellular levels of cholesterol (a) and triglyceride (b) were extracted by hexane/isopropanol (3/2, v/v) and analyzed by colorimetric assay kits or (c) subjected to oil-red O staining. Cellular nuclei were stained with hematoxylin. Data is mean ± SEM from 5 independent experiments. ∗ P < 0.05 versus vehicle-treated group, # P < 0.05 versus oxLDL alone-treated group, $ P < 0.05 versus oxLDL+EPO-treated group.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Colorimetric Assay, Staining

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: Knockdown of β CR expression ablates the EPO-mediated protection of oxLDL-induced lipid accumulation in macrophages. (a) Bone-marrow-derived macrophages were transfected with indicated concentrations of β CR siRNA (25, 50, and 100 nmole/L) for 24 h. Protein level of β CR was examined by western blot analysis. Cells were transfected with control siRNA (50 nmole/L) or β CR siRNA (50 nmole/L) for 24 h and then treated with EPO (5 U/mL) for 12 h, followed by oxLDL (50 μ g/mL) for additional 18 h. ((b) and (c)) The intracellular levels of cholesterol and triglyceride were extracted by hexane/isopropanol (3/2, v/v) and analyzed by colorimetric assay kits or (d) subjected to oil-red O staining. Cellular nuclei were stained with hematoxylin. Data is mean ± SEM from 5 independent experiments. ∗ P < 0.05 versus vehicle-treated group, # P < 0.05 versus oxLDL alone-treated group, $ P < 0.05 versus oxLDL+EPO-treated group.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Derivative Assay, Transfection, Western Blot, Control, Colorimetric Assay, Staining

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: Inhibition of β CR activation abolishes the EPO-mediated promotion of cholesterol efflux in macrophages. Bone-marrow-derived macrophages were pretreated with IgG (200 ng/mL) or β CR neutralizing Ab (200 ng/mL) for 2 h (b) transfection with control siRNA (50 nmole/L) or β CR siRNA (50 nmole/L) for 24 h and then treated with EPO (5 U/mL) for 12 h, followed by NBD-cholesterol (1 μ g/mL) for additional 6 h in the absence or presence of apoAI (10 μ g/mL) or HDL (50 μ g/mL). The medium and cell lysates were collected for the measurement of fluorescence. The calculation of cholesterol efflux is defined as fluorescence in the medium relative to the total amount of fluorescence. Data are mean ± SEM from 5 independent experiments. ∗ P < 0.05 versus vehicle-treated group, # P < 0.05 versus EPO-treated group.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Activation Assay, Derivative Assay, Transfection, Control, Fluorescence

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: Blockage of β CR activation ablates EPO-induced upregulation of ABCA1 and ABCG1 transporters in macrophages. Bone-marrow-derived macrophages were pretreated with IgG (200 ng/mL) or β CR neutralizing Ab (200 ng/mL) for 2 h (b) transfection with control siRNA (50 nmole/L) or β CR siRNA (50 nmole/L) for 24 h and then treated with EPO (5 U/mL) for 12 h or 24 h. Cell were lyzed and lyates subjected to RT-PCR and western blot analysis for mRNA or protein expression of ABCA1, ABCG1, and GAPDH. Data is mean ± SEM from 5 independent experiments. ∗ P < 0.05 versus vehicle-treated group and # P < 0.05 versus EPO-treated group.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Derivative Assay, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: Mediators of Inflammation

Article Title: β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

doi: 10.1155/2015/439759

Figure Lengend Snippet: Inhibition of β CR activation prevents the protective effect of EPO against from the oxLDL-induced MIP-2 production in macrophages. (a) BMDMs were treated with indicated concentrations of oxLDL (12.5, 25, 50, and 100 μ g/mL) for 24 h. (b) Cells were treated with oxLDL (50 μ g/mL) in the absence or presence of indicated concentrations of EPO (2.5, 5, and 10 U/mL) for 24 h. (c) Cells were pretreated with IgG (200 ng/mL) or β CR neutralizing Ab (200 ng/mL) for 2 h or (d) transfection with control siRNA (50 nmole/L) or β CR siRNA (50 nmole/L) for 24 h and then treated with oxLDL (50 μ g/mL) and EPO (5 U/mL) for an additional 24 h. Levels of MIP-2 in cultured medium were analyzed by ELISA. Data are mean ± SD from 5 independent experiments. ∗ P < 0.05 versus vehicle-treated group, # P < 0.05 versus oxLDL alone-treated group, and $ P < 0.05 versus oxLDL-treated group with EPO-treatment.

Article Snippet: Goat anti-EPO, rabbit anti-EPOR, anti- β CR antibodies, control small interfering RNA (siRNA), and β CR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Activation Assay, Transfection, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Knockdown of EZH2 promotes differentiation of HL-60 AML cells by ATRA. (A) Expression of the myeloid differentiation marker CD11b (left panel), and proportion of live cells (PI-negative population, right panel) following lentivirally-mediated knockdown (KD) of EZH2 expression and analysis by flow cytometry. EZH2 KD was achieved using two different short-hairpin RNA sequences (shRNA #1 and #2) as indicated. A non-targeting shRNA was used as a negative control. Values represent the means of three experiments and error bars denote standard deviations. * p < 0.05; *** p < 0.001; ns, no statistical significance. (B) Immunoblot analysis of levels of EZH2 protein and trimethylated H3 Lys27 (H3K27 me3 ) following EZH2 KD. Transduced cells were selected with puromycin and expanded for a minimum of 10 days. GAPDH was used as a loading control. (C) Cell morphology of cells analyzed by May-Grunwald Giemsa staining following EZH2 KD (shRNA #1) +/− ATRA (0.1 µM for 72 h).

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Marker, Flow Cytometry, shRNA, Negative Control, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Dual inhibition of EZH2-G9A/GLP by HKMTI-1-005 promotes differentiation of HL-60 AML cells by ATRA. (A) Proportion of viable cells as determined by CellTiter-Glo cell viability assay (left panel), and expression of the myeloid differentiation marker CD11b (right panel) following treatment with the indicated concentrations of GSK-343 +/− 0.1 µM ATRA for 72 h. (B) Proportion of viable cells as determined by CellTiter-Glo cell viability assay (left panel) and expression of the myeloid differentiation marker CD11b (right panel) following treatment with the indicated concentrations of HKMTI-1-005 +/− 0.1 µM ATRA for 72 h. Values represent the means of three experiments (viability assays) or two experiments (CD11b flow cytometry). Error bars denote standard deviations. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no statistical significance. (C) Cell morphology of cells analyzed by May-Grunwald Giemsa staining following treatment with 10 µM GSK-343 (left panel) or 2.5 µM HKMTI-1-005 (right panel) for 72 h. Treatments were performed +/− 0.1 µM ATRA. (D) Immunoblot analysis of levels of trimethylated H3 Lys27 (H3K27 me3 ), dimethylated H3 Lys9 (H3K9 me2 ) and trimethylated H3 Lys9 (H3K9 me3 ) following treatment (72 h) with 10 µM GSK-343 or 2.5 µM HKMTI-1-005 +/− 0.1 µM ATRA as indicated. GAPDH was used as a loading control.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Inhibition, Viability Assay, Expressing, Marker, Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Differential gene expression following EZH2 knockdown or inhibition with GSK-343 or HKMTI-1-005 in HL-60 AML cells. (A) Principal component analysis (PCA; covariance) of GCRMA-normalized expression array samples in biological triplicates represented with centroids as annotated: two different EZH2 -targeting shRNAs and one non-targeting control (days ∼10–20 post transduction), 10 µM GSK-343 or 2.5 µM HKMTI-1-005 +/− 0.1 µM ATRA following treatment for 72 h. Red or blue ellipses highlight specific grouping of samples of interest. (B) Gene ontology (GO) analysis of upregulated genes following EZH2 KD or drug treatment as annotated (0.1 µM ATRA, 10 µM GSK-343, 2.5 µM HKMTI-1-005 for 72 h) showing enrichment score (left panel) and fold-enrichment over control (right panel) of genes belonging to the indicated categories. (C) Venn diagram showing groups of genes taken for further analysis that belong exclusively to GSK-343 treated cells, or are shared only between EZH2 KD and HKMTI-1-005 treated cells, or between EZH2 KD, HKMTI-1-005 and ATRA, but not GSK-343 treated cells. Gene groups are shaded grey and indicated.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Inhibition, Transduction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of gene expression in HL-60 cells reveals opposite effects of GSK-343 and HKMTI-1-005 on stem cell and differentiation programs. Gene expression data were generated by expression microarray analysis following 72 h treatment with 10 µM GSK-343 or 2.5 µM HKMTI-1-005, or EZH2 KD versus untreated control and analyzed using GSEA to extract biological knowledge. Highly significantly enriched gene sets are indicated by their standard and systematic names. The most upregulated genes in treatment or EZH2 KD samples are shown on the left side (red), while the most upregulated genes in the control are shown on the right side (blue). Black bars represent the positions of the treatment or EZH2 KD samples versus vehicle control upregulated signature genes in the ranked list. Green curves represent the evolution of gene density. Normalized enrichment scores (NES) reflect the degree to which genes were overrepresented. When the distribution is random, the enrichment score is zero. Enrichment of signature genes at the top of the ranked list results in a large positive deviation of the NES from zero. q -value = FDR-adjusted p -value.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Generated, Microarray

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: ATRA-mediated differentiation promotes an association between RARα and EZH2 in HL-60 cells that is inhibited by GSK-343 and enhanced by HKMTI-1-005. (A) Proximity ligation assay (PLA) analyzing RARα/EZH2 complexes following 0.1 µM ATRA treatment for the indicated times. (B) PLA analyzing RARα/EZH2 complexes following treatment with 10 µM GSK-343 or 2.5 µM HKMTI-1-005 for 4 h. Treatments were performed +/− 0.1 µM ATRA as indicated. Upper panels show representative fields of view of cells indicating proximity (< 40 nm) of antibody conjugated PLA probes that have been ligated, amplified, and detected with complementary fluorescent probes. Red dots represent the presence of RARα/EZH2 interactions. Scale bars, 20 µm. Cell nuclei are counterstained with DAPI (blue). Lower panels show mean values of signals (red dots) per cell representing RARα/EZH2 interactions. Values represent the means of two experiments (A) or three experiments (B) , and error bars denote standard deviations. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no statistical significance.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Proximity Ligation Assay, Amplification

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Knockdown of EZH2 promotes differentiation of HL-60 AML cells by ATRA. (A) Expression of the myeloid differentiation marker CD11b (left panel), and proportion of live cells (PI-negative population, right panel) following lentivirally-mediated knockdown (KD) of EZH2 expression and analysis by flow cytometry. EZH2 KD was achieved using two different short-hairpin RNA sequences (shRNA #1 and #2) as indicated. A non-targeting shRNA was used as a negative control. Values represent the means of three experiments and error bars denote standard deviations. * p < 0.05; *** p < 0.001; ns, no statistical significance. (B) Immunoblot analysis of levels of EZH2 protein and trimethylated H3 Lys27 (H3K27 me3 ) following EZH2 KD. Transduced cells were selected with puromycin and expanded for a minimum of 10 days. GAPDH was used as a loading control. (C) Cell morphology of cells analyzed by May-Grunwald Giemsa staining following EZH2 KD (shRNA #1) +/− ATRA (0.1 µM for 72 h).

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Marker, Flow Cytometry, shRNA, Negative Control, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Dual inhibition of EZH2-G9A/GLP by HKMTI-1-005 promotes differentiation of HL-60 AML cells by ATRA. (A) Proportion of viable cells as determined by CellTiter-Glo cell viability assay (left panel), and expression of the myeloid differentiation marker CD11b (right panel) following treatment with the indicated concentrations of GSK-343 +/− 0.1 µM ATRA for 72 h. (B) Proportion of viable cells as determined by CellTiter-Glo cell viability assay (left panel) and expression of the myeloid differentiation marker CD11b (right panel) following treatment with the indicated concentrations of HKMTI-1-005 +/− 0.1 µM ATRA for 72 h. Values represent the means of three experiments (viability assays) or two experiments (CD11b flow cytometry). Error bars denote standard deviations. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no statistical significance. (C) Cell morphology of cells analyzed by May-Grunwald Giemsa staining following treatment with 10 µM GSK-343 (left panel) or 2.5 µM HKMTI-1-005 (right panel) for 72 h. Treatments were performed +/− 0.1 µM ATRA. (D) Immunoblot analysis of levels of trimethylated H3 Lys27 (H3K27 me3 ), dimethylated H3 Lys9 (H3K9 me2 ) and trimethylated H3 Lys9 (H3K9 me3 ) following treatment (72 h) with 10 µM GSK-343 or 2.5 µM HKMTI-1-005 +/− 0.1 µM ATRA as indicated. GAPDH was used as a loading control.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Inhibition, Viability Assay, Expressing, Marker, Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Differential gene expression following EZH2 knockdown or inhibition with GSK-343 or HKMTI-1-005 in HL-60 AML cells. (A) Principal component analysis (PCA; covariance) of GCRMA-normalized expression array samples in biological triplicates represented with centroids as annotated: two different EZH2 -targeting shRNAs and one non-targeting control (days ∼10–20 post transduction), 10 µM GSK-343 or 2.5 µM HKMTI-1-005 +/− 0.1 µM ATRA following treatment for 72 h. Red or blue ellipses highlight specific grouping of samples of interest. (B) Gene ontology (GO) analysis of upregulated genes following EZH2 KD or drug treatment as annotated (0.1 µM ATRA, 10 µM GSK-343, 2.5 µM HKMTI-1-005 for 72 h) showing enrichment score (left panel) and fold-enrichment over control (right panel) of genes belonging to the indicated categories. (C) Venn diagram showing groups of genes taken for further analysis that belong exclusively to GSK-343 treated cells, or are shared only between EZH2 KD and HKMTI-1-005 treated cells, or between EZH2 KD, HKMTI-1-005 and ATRA, but not GSK-343 treated cells. Gene groups are shaded grey and indicated.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Inhibition, Transduction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of gene expression in HL-60 cells reveals opposite effects of GSK-343 and HKMTI-1-005 on stem cell and differentiation programs. Gene expression data were generated by expression microarray analysis following 72 h treatment with 10 µM GSK-343 or 2.5 µM HKMTI-1-005, or EZH2 KD versus untreated control and analyzed using GSEA to extract biological knowledge. Highly significantly enriched gene sets are indicated by their standard and systematic names. The most upregulated genes in treatment or EZH2 KD samples are shown on the left side (red), while the most upregulated genes in the control are shown on the right side (blue). Black bars represent the positions of the treatment or EZH2 KD samples versus vehicle control upregulated signature genes in the ranked list. Green curves represent the evolution of gene density. Normalized enrichment scores (NES) reflect the degree to which genes were overrepresented. When the distribution is random, the enrichment score is zero. Enrichment of signature genes at the top of the ranked list results in a large positive deviation of the NES from zero. q -value = FDR-adjusted p -value.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Expressing, Generated, Microarray

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells

doi: 10.3389/fcell.2023.1076458

Figure Lengend Snippet: ATRA-mediated differentiation promotes an association between RARα and EZH2 in HL-60 cells that is inhibited by GSK-343 and enhanced by HKMTI-1-005. (A) Proximity ligation assay (PLA) analyzing RARα/EZH2 complexes following 0.1 µM ATRA treatment for the indicated times. (B) PLA analyzing RARα/EZH2 complexes following treatment with 10 µM GSK-343 or 2.5 µM HKMTI-1-005 for 4 h. Treatments were performed +/− 0.1 µM ATRA as indicated. Upper panels show representative fields of view of cells indicating proximity (< 40 nm) of antibody conjugated PLA probes that have been ligated, amplified, and detected with complementary fluorescent probes. Red dots represent the presence of RARα/EZH2 interactions. Scale bars, 20 µm. Cell nuclei are counterstained with DAPI (blue). Lower panels show mean values of signals (red dots) per cell representing RARα/EZH2 interactions. Values represent the means of two experiments (A) or three experiments (B) , and error bars denote standard deviations. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no statistical significance.

Article Snippet: Antibodies used for immunoblot and immunoprecipitation were as follows: rabbit polyclonal anti-H3K27 me3 (Cat# 07–473, Upstate Biotechnology); rabbit polyclonal anti-H3K9 me2 (Cat# ab115159, Abcam); rabbit polyclonal anti-GAPDH (Cat# ab9485, Abcam); rabbit polyclonal anti-EZH2 (Cat# ab186006, Abcam); mouse monoclonal anti-EZH2 (clone AC22, Cat# 3147, Cell Signaling Technology); mouse monoclonal anti-RARα (clone 9α-9A6, Cat# 39971, Active Motif); rabbit polyclonal anti-RARα (Cat# sc-773, Santa Cruz).

Techniques: Proximity Ligation Assay, Amplification