Journal: Breast Cancer Research : BCR

Article Title: BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

doi: 10.1186/s13058-018-1046-3

Figure Lengend Snippet: BHLHE40 activates gene expression by sequestering histone deacetylase (HDAC)1 and HDAC2 from genome DNA binding in MDA-MB-231 cells exposed to hypoxia and low glucose (1%O 2 /LG, 4 h). a BHLHE40-knockout (KO) diminished dissociation of HDAC1 and HDAC2 from the promoter region of HBEGF in MDA-MB-231 cells exposed to 1%O 2 /LG (4 h), as determined by chromatin immunoprecipitation (ChIP) followed by qPCR of the HBEGF promoter region (−529 to −372 from the transcription start site). HBEGF promoter binding activity of HDAC1 or HDAC2 was calculated as: (DNA amount in anti-HADC IP complex – DNA amount in control IgG IP complex)/DNA amount in 1% input. * p < 0.05 ( n = 6, 1%O 2 /LG vs. control), ** p < 0.05 ( n = 6, KO vs. EV). b 1%O 2 /LG treatment increased interactions between BHLHE40 and HDAC1/2 in the soluble cellular fraction of MDA-MB-231 empty vector (EV) cells. Protein-protein interaction was detected by reciprocal co-immunoprecipitation (IP)/immunoblotting (IB) analysis. c HDAC inhibition induced expression of BHLHE40 target genes. Cells were exposed to hypoxia (1% O 2 ) or HDAC inhibitors (BRD6688 10 μM or TSA 2 μM) for 24 h. mRNA expression levels were determined by qPCR, normalized to RPL13A, and presented as mean ± SD ( n = 6). * p < 0.05 ( n = 6, treated vs. untreated control cells), one-way ANOVA followed by Tukey’s post-hoc tests

Article Snippet: To detect protein-protein interaction, soluble proteins were extracted using the Pierce IP Lysis Buffer (Thermo Scientific) supplemented with protease inhibitor cocktails and co-immunoprecipitation (CoIP) was performed using the TrueBlot Immunoprecipitation and Western Blot Kit (Rockland Immunochemicals Inc., Limerick, PA, USA).

Techniques: Gene Expression, Histone Deacetylase Assay, Binding Assay, Knock-Out, Chromatin Immunoprecipitation, Activity Assay, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Inhibition, Expressing