Journal: Molecular Cancer

Article Title: Epigenetic reactivation of estrogen receptor-α (ERα) by genistein enhances hormonal therapy sensitivity in ERα-negative breast cancer

doi: 10.1186/1476-4598-12-9

Figure Lengend Snippet: Epigenetic alterations in response to GE and/or TSA treatments. ( A ) Histone modification patterns in the ERα promoter were analyzed by ChIP assay. Representative photograph from an experiment was repeated in triplicate. ( B ) Histone modification enrichment in the ERα promoter was calculated from the corresponding DNA fragments amplified by ChIP-PCR as shown above. MDA-MB- 231 cells were treated as described previously and analyzed by ChIP assays using chromatin markers including acetyl-H3, acetyl-H3K9, acetyl-H4, dimethyl-H3K4 and mouse IgG control in the promoter region of ERα . Inputs came from the total DNA and served as the same ChIP-PCR conditions. DNA enrichment was calculated as the ratio of each bound sample divided by the input while the untreated MDA-MB-231 control sample is represented as 1. ( C ) HDACs enzymatic activity. ( D ) DNMTs enzymatic activity. Nuclear proteins of MDA-MB-231 cells were extracted after the treatment as described above. The HDACs and DNMTs activity assays were performed according to the manufacturer’s protocols. ( E ) Binding abilities of HDACs and DNMTs in the ERα promoter were determined by ChIP assay as described previously. The values of enzymatic activities of HDACs and DNMTs are the means of three independent experiments. Columns, mean; Bars, SD. *, P < 0.05, significantly different from control; £, P < 0.05, significantly different from GE; †, P < 0.05, significantly different from TSA. ( F ) The protein level changes of HDACs and DNMTs were determined by western-blot analysis. GAPDH antibody was used to ensure equal loading. Representative photograph from an experiment was repeated three times.

Article Snippet: The activities of HDACs (Active Motif, Carlsbad, CA) and DNMTs (Epigentek, Brooklyn, NY, USA) were performed according to the manufacturer’s protocols as reported previously [ , ].

Techniques: Modification, Amplification, Control, Activity Assay, Binding Assay, Western Blot

Journal: Molecular Cancer

Article Title: Epigenetic reactivation of estrogen receptor-α (ERα) by genistein enhances hormonal therapy sensitivity in ERα-negative breast cancer

doi: 10.1186/1476-4598-12-9

Figure Lengend Snippet: Epigenetic enzymatic activities in response to dietary GE and/or TAM treatment in mice breast tumors. Left panel, MDA-MB-231 breast tumor xenografts ( Protocol 1 ); right panel, C3(1)-SV40 Tag transgenic mouse tumors ( Protocol 2 ). GE and/or TAM treatment were described above. A ) HDACs enzymatic activity. B ) DNMTs enzymatic activity. Nuclear proteins from mice breast tumors were extracted as described previously. The HDACs and DNMTs activity assays were performed according to the manufacturer’s protocols. The values of enzymatic activities of HDACs and DNMTs are the means of three independent experiments from all tumors per group. Columns, mean; Bars, SD. *, P < 0.05, significantly different from control; * * P < 0.01, significantly different from control.

Article Snippet: The activities of HDACs (Active Motif, Carlsbad, CA) and DNMTs (Epigentek, Brooklyn, NY, USA) were performed according to the manufacturer’s protocols as reported previously [ , ].

Techniques: Transgenic Assay, Activity Assay, Control

Journal: PLoS ONE

Article Title: Inhibition of Histone Deacetylases 1 and 6 Enhances Cytarabine-Induced Apoptosis in Pediatric Acute Myeloid Leukemia Cells

doi: 10.1371/journal.pone.0017138

Figure Lengend Snippet: Panel A: THP-1 cells were treated with equal doses (IC 20 s, determined by MTT assays) of MS-275, VPA or SAHA for 48 hrs. Acetylation of histones H3 and H4, and α-tubulin were determined by western blots probed by anti-ac-H3, -ac-H4, -ac-tubulin, or –H4 antibodies. Panel B: THP-1 cells were treated with cytarabine (900 nM, IC 20 ) or equal doses of MS-275, VPA, or SAHA, alone or in combination for 48 hrs. Early and late apoptosis events were determined by annexin V/PI staining and flow cytometry analysis. **, p <0.005; *, p <0.05. Panels C&D: THP-1 cells were treated with equal doses (IC 20 s) of MS-275, VPA or SAHA for 48 hrs and protein extracts were subjected to immunoprecipitation with antibodies against class I HDACs, as described in the . One half of the immunoprecipitated proteins was subjected to Western blots probed by anti-HDAC1, -HDAC2, -HDAC3, or -HDAC8 antibody (panel C), and the other half was used for measuring class I HDAC activities (panel D). The data are presented as means of three independent experiments normalized to the non-drug treatment controls.

Article Snippet: The beads were washed three times with ice cold PBS and resuspended in HDAC Assay Buffer [40 μL; 20 mmol/L Tris-HCl (pH 8), 125 mmol/L NaCl, and 1% glycerol] for measuring HDAC enzymatic activities using the CycLex® HDACs Deacetylase Fluorometric Assay kit (CycLex, Nagano, Japan), or heated at 95°C for 5 min in 30 μl loading buffer for Western blotting.

Techniques: Western Blot, Staining, Flow Cytometry, Immunoprecipitation

Journal: Molecular Cancer

Article Title: Synergistic epigenetic reactivation of estrogen receptor-α (ERα) by combined green tea polyphenol and histone deacetylase inhibitor in ERα-negative breast cancer cells

doi: 10.1186/1476-4598-9-274

Figure Lengend Snippet: Alteration of histone modulation of the ERα promoter and histone acetylation-related enzymatic activities . A) Histone modification patterns were analyzed by ChIP assay. Representative photograph from an experiment was repeated in triplicate. B) Histone modification enrichment in the ERα promoter was calculated from the corresponding DNA fragments amplified by ChIP-PCR as shown above. MDA-MB-231 cells were treated as described previously and analyzed by ChIP assays using chromatin markers including acetyl-H3, acetyl-H3K9, acetyl-H4, dimethyl-H3K4, trimethyl-H3K9 and mouse IgG control in the promoter region of ERα. MCF-7 cells served as a positive control. Inputs came from the total DNA and served as the same ChIP-PCR conditions. DNA enrichment was calculated as the ratio of each bound sample divided by the input while the untreated MDA-MB-231 control sample is represented as 1.0. Columns, mean; Bars, SD. C) and D) Histone acetyltransferase (HATs) and Histone deacetylases (HDACs) activities in MDA-MB-231 cells. Nuclear proteins of MDA-MB-231 cells were extracted after the treatment as described above. The HATs and HDACs activity assays were performed according to the manufacturer's protocols. The values of enzymatic activities of HATs and HDACs are the means of three independent experiments. Columns, mean; Bars, SD; *, P < 0.05, * * P < 0.001, significantly different from control.

Article Snippet: The activities of histone deacetylases, HDACs (Active Motif, Carlsbad, CA), and histone acetyltransferases, HATs (Epigentek, Brooklyn, NY), were performed according to the manufacturer's protocols as reported previously [ ].

Techniques: Modification, Amplification, Control, Positive Control, Activity Assay