Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Chromatin immunoprecipitation assay for histone acetylation (acH3, acH4), H3K4 trimethylation (H3K4me3) and histone H3K9 monomethylation (H3K9me) at the 5-LO core promoter in Mono Mac6 knockdown cell lines. Cells were grown with or without TSA (330 nM) for 24 hrs. Then, the cells were harvested and subjected to ChIP analysis. Thirty-two PCR cycles were applied.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Chromatin immunoprecipitation assay for histone H3K4 trimethylation (H3K4me3) and histone H3K9 monomethylation (H3K9me) at the 5-LO core promoter in Mono Mac6, HL-60 and U937 cells. Cells were grown with TSA (330 nM) for the indicated times. Then, the cells were harvested and subjected to ChIP analysis. Thirty-two PCR cycles were applied.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Time course of 5-LO mRNA induction by TSA in Mono Mac6, HL-60 and U937 cells. The cells were cultured in the presence of TSA (330 nM) with or without CHX (50 μM) for the indicated times. Then, the cells were harvested and RNA was isolated, reverse transcribed into cDNA and analysed by quantitative real-time PCR for 5-LO mRNA expression. Data are shown as mean ± S.E. from three independent experiments.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Effects of HDAC 1, 2 and 3 knockdown on 5-LO mRNA expression in Mono Mac6 cells. (A) 5-LO mRNA expression in Mono Mac6 cells and the respective Mono Mac6 HDAC knockdown cell lines was determined by quantitative real-time PCR. Results are given as 5-LO mRNA copy number per 10 6 β-actin mRNA copies. Data are shown as mean + S.E. of six independent experiments. (B) Western blot analysis of HDAC expression in the respective HDAC knockdown cell lines. A total of 6 χ 10 6 cells were harvested and analysed by Western blot for the indicated HDAC isoform.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Effects of HDAC inhibitors (TSA, apicidin and MS-275) on 5-LO mRNA expression in HDAC1, HDAC2 and HDAC3 knockdown cell lines of Mono Mac6. HDAC1 (A), HDAC2 (B) and HDAC3 (C) knockdown cells were treated for 24 hrs with the indicated HDAC inhibitors at the indicated concentrations. Then, 5-LO mRNA expression in the respective Mono Mac6 HDAC knockdown cell lines was determined by quantitative real-time PCR. Results are given as 5-LO mRNA copy number per 10 6 β-actin mRNA copies. Data are shown as mean + S.E. of three independent experiments.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Mass Spectrometry, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Chromatin immunoprecipitation assay (ChIP) for acetylated histones H3 (acH3) and H4 (acH4) at the 5-LO core promoter in U937, HL-60 and Mono Mac6 cells. Normal rabbit IgG antibodies served as negative control to detect unspecific binding to histone proteins and the no antibody control (no ab) was used to detect unspecific binding to the immunoprecipitation beads. The input sample (sheared chromatin without immunoprecipitation) served as positive control for the PCR reaction. (A) Analysis of the acetylation status of the histone proteins H3 and H4 at the 5-LO promoter in U937, HL-60 and Mono Mac6 cells. 32 PCR cycles were applied. (B) Time course of H3 and H4 acetylation of the 5-LO promoter in the indicated cell lines. Cells were treated with TSA (330 nM) for the indicated times. Then, the cells were harvested and analysed by ChIP as described. Thirty-two PCR cycles were performed except for AcH4 in Mono Mac6 cells, where the cycle number was reduced to 28.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Immunoprecipitation, Positive Control, Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

doi: 10.1111/j.1582-4934.2011.01420.x

Figure Lengend Snippet: Up-regulation of 5-LO promoter activity (A) and 5-LO mRNA expression (B) by histone deacetylase inhibitors. (A) 5-LO promoter activity was determined by reporter gene assays. HeLa cells were transfected with the 5-LO promoter construct pN10 (comprises bp –844 to –12 in relation to ATG) and cultured in the presence of the indicated HDAC inhibitors for 24 hrs. Then, reporter gene activity was determined. Results are expressed as relative light units (RLU) of the internal standard plasmid pCMVRenilla and are shown as mean + S.E. ( n = 3). (B) Real-time PCR analysis of 5-LO mRNA expression in Mono Mac6 cells. Cells were treated with the indicated HDAC inhibitors for 24 hrs. Then, the cells were harvested, RNA was isolated, reverse transcribed into cDNA and 5-LO mRNA expression was determined by real-time PCR. Values are given as the mean + S.E. of three independent experiments.

Article Snippet: Mono Mac6 cells (DSMZ, ACC 124) were grown in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1χ non-essential amino acids, sodium pyruvate (1 mM), oxalacetate (1 mM), insulin (10 μg/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Activity Assay, Expressing, Histone Deacetylase Assay, Transfection, Construct, Cell Culture, Plasmid Preparation, Real-time Polymerase Chain Reaction, Isolation