Fig. 6
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%O2/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%O2/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%O2/LG vs. control), **p <  0.05 (n = 6, KO vs. EV). b 1%O2/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% O2) 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