Journal: Oncogene

Article Title: Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

doi: 10.1038/onc.2016.186

Figure Lengend Snippet: HDAC5 stabilizes LSD1 protein in breast cancer cells. (a) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 48 h. mRNA expression of HDAC5 and LSD1 was measured by quantitative real-time PCR with β-actin as an internal control. (b) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h. Effect of HDAC5 overexpression on LSD1 protein expression in MDA-MB-231 cells was evaluated by immunoblots with anti-LSD1 and anti-HDAC5 antibodies. (c) MDA-MB-231 cells were transfected with scramble siRNA or HDAC5 siRNA for 48 h. Effect of HDAC5 knockdown on LSD1 mRNA expression was examined by quantitative real-time PCR with β-actin as internal control. (d) Effect of HDAC5 siRNA on LSD1 protein expression in MDA-MB-231 cells. (e) Effect of depletion of LSD1 on mRNA expression of HDAC5 in MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells. (f) Effect of LSD1-KD on protein expression of HDAC5 in MDA-MB-231-scramble or MDA-MB-231-LSD1-KD cells. (g) MDA-MB-231 cells were transfected with control vector pcDNA3.1, pcDNA3.1-HDAC5, scramble siRNA or HDAC5 siRNA for 48 h and analyzed by immunoblots for nuclear expression of indicated histone marks. PCNA was used as loading control. (h) Effect of HDAC5 overexpression or siRNA on LSD1 protein half-life in cycloheximide chase study. (i) Measurement of LSD1 half-life using Calcusyn program. (j) Effect of siRNA knockdown of LSD1 cofactors or class II HDACs on LSD1 protein level. All the experiments were performed three times. Bars represent the mean of three independent experiments ± s.d. * p <0.05, ** p <0.01, *** p <0.001, Student’s t-test.

Article Snippet: Scramble control, LSD1-specific or HDAC5-specific shRNA lentiviral particles (Santa Cruz) were infected into cells according to manufacturer’s protocol.

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

Journal: Oncogene

Article Title: Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

doi: 10.1038/onc.2016.186

Figure Lengend Snippet: HDAC5 regulates LSD1 by altering USP28 stability. (a) MDA-MB-231 cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-HDAC5 or pcDNA3-HA-ubiquitin plasmids were treated with or without proteasome inhibitor 10µM MG132 for 10 h followed by immunoprecipitation (IP) using LSD1 antibody and immunoblots with anti-HA, LSD1 or HDAC5 antibodies. (b) Effect of siRNA of Jade-2, USP28 and HDAC5 on LSD1 protein expression in MDA-MB-231 cells. Results represent the mean of three independent experiments ± s.d. *** p <0.001, Student’s t-test. (c) MDA-MB-231 cells were transfected with scramble siRNA, HDAC5-siRNA, control vector pcDNA3.1, or pcDNA3.1-HDAC5 plasmids for 48 h. mRNA expression of Jade-2 and USP28 was measured by quantitative PCR. β-actin was used as an internal control. (d) MDA-MB-231 or MCF10A–CA1a cells were simultaneously transfected with pcDNA3.1-FLAG-Jade-2 and HDAC5 siRNA for 48 h and subjected to immunoblots with anti-HDAC5 or Jade-2 antibodies. β-actin was used as loading control to normalize target protein levels. (e) After MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h, immunoblotting was performed for expression of HDAC5 and USP28. (f) MDA-MB-231 or MCF10A–CA1a cells were transfected with scramble or HDAC5 siRNA alone, or in combination with pDZ-USP28 for 48 h. Whole cell lysates were analyzed for protein levels of HDAC5, USP28 and LSD1. β-actin was used as loading control to normalize target protein levels. The experiments were performed three times with similar results.

Article Snippet: Scramble control, LSD1-specific or HDAC5-specific shRNA lentiviral particles (Santa Cruz) were infected into cells according to manufacturer’s protocol.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

doi: 10.1038/onc.2016.186

Figure Lengend Snippet: Effect of HDAC5 on protein acetylation of LSD1/USP28 and transcription of LSD1 target genes. (a) The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tagged USP28 or FLAG-tagged LSD1 were used as substrates for protein deacetylation assay. IgG was used as negative control. Active or heat inactivated recombinant human GST-tagged HDAC5 protein were mixed with immunoprecipitates and incubated at 37°C for 6 h as described in “ Materials and Methods ”. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody. FLAG-tagged USP28 or LSD1 proteins were probed with anti-FLAG antibody. HDAC5-GST protein was probed with anti-HDAC5 antibody. (b) Histograms represent the means of levels of acetyl-LSD1, acetyl-USP28 and acetyl-histone determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (c) MDA-MB-231 cell transfected with scramble or HDAC5 siRNAs for 48 h. LSD1 or IgG antibodies were added to cell lysate. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting with anti-acetyl lysine and anti-LSD1 antibodies, respectively. Effect of HDAC5 siRNA on Acetyl-H3K9 protein expression in MDA-MB-231 cells was examined by immunoblotting with anti-acetyl-H3K9 antibody. (d) Histograms represent the means of relative levels of acetyl-LSD1 determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (e) mRNA expression of indicated genes in MDA-MB-231 cells transfected with scramble siRNA or HDAC5 siRNA. Data are means ± s.d. of three independent experiments. (f) Quantitative ChIP analysis was used to determine the occupancy by acetyl-H3K9, H3K4me2, LSD1, and HDAC5 at promoters of p21 or CLDN7 in MDA-MB-231 cells transfected with scramble or HDAC5 siRNA. * p<0.05 , ** p<0.01 , *** p<0.001 , Student’s t-test.

Article Snippet: Scramble control, LSD1-specific or HDAC5-specific shRNA lentiviral particles (Santa Cruz) were infected into cells according to manufacturer’s protocol.

Techniques: Negative Control, Recombinant, Incubation, Western Blot, Transfection, Immunoprecipitation, Expressing

Journal: Oncogene

Article Title: Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

doi: 10.1038/onc.2016.186

Figure Lengend Snippet: HDAC5-LSD1 axis is implicated in breast cancer progression. (a) Depletion of HDAC5 by shRNA lentivirus infection downregulated LSD1 protein expression in MDA-MB-231 and MCF10A–CA1a cells. (b) Scramble shRNA and HDAC5-KD cells were analyzed for growth and viability by crystal violet assays. (c) Soft agar colony formation for HDAC5-KD and scramble control of MDA-MB-231 and MCF10A–CA1a cells. (d) Scramble shRNA and HDAC5-KD cells were harvested and stained for DNA with propidium iodide for flow cytometric analysis. The fractions corresponding to G1, S and G2/M phases of the cell cycle are indicated. (e) Boyden Chamber transwell migration assays for cell invasion for MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD-1 cells. (f) MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 5 days and crystal violet assays for growth were carried out. (g) MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD cells were transfected with empty or pReceiver-LSD1 expression plasmids for 5 days and crystal violet assays for growth were carried out. Bars represent the means of three independent experiments ± s.d. * p <0.05, ** p <0.01, *** p <0.001, Student’s t-test.

Article Snippet: Scramble control, LSD1-specific or HDAC5-specific shRNA lentiviral particles (Santa Cruz) were infected into cells according to manufacturer’s protocol.

Techniques: shRNA, Infection, Expressing, Control, Staining, Migration, Transfection, Plasmid Preparation

Journal: Oncogene

Article Title: Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

doi: 10.1038/onc.2016.186

Figure Lengend Snippet: Effect of HDAC5 on growth and mutagen-induced tumorigenic transformation in MCF10A cells. (a) pcDNA3.1 or pcDNA3.1-HDAC5 transfected MCF10A cells (clone 1 and 2) were analyzed for protein levels of HDAC5 and LSD1 by immunoblots with anti-HDAC5 and anti-LSD1 antibodies. (b) Crystal violet assay for growth of MCF10-A stably transfected with control vector or pcDNA3.1-HDAC5 plasmids. (c) MCF10A–Vector-1 or MCF10A–HDAC5-1 cells were infected with scramble or LSD1 shRNA lentivirus particles for 5 days followed by crystal violet assays for growth. (d) MCF10A cells transfected with pcDNA3.1 or pcDNA3.1-HDAC5 plasmids were treated with DMSO or 500 ng/ml ICR191 for 7 months followed by soft agar colony formation assays. (e) After treatment with 500 ng/ml ICR191 for 7 months, MCF10A–HDAC5 cells were infected with scramble control or LSD1 shRNA lentivirus particles and soft agar colony formation assay was carried out. (f) Proposed model of the role of HDAC5-LSD1 axis in breast cancer development. Bars represent the means of three independent experiments ± s.d. ** p <0.01, *** p <0.001, Student’s t-test.

Article Snippet: Scramble control, LSD1-specific or HDAC5-specific shRNA lentiviral particles (Santa Cruz) were infected into cells according to manufacturer’s protocol.

Techniques: Transformation Assay, Transfection, Western Blot, Crystal Violet Assay, Stable Transfection, Control, Plasmid Preparation, Infection, shRNA, Soft Agar Assay