Fig. 4
BHLHE40-knockdown reduced hypoxia-induced expression of a panel of cytokines and growth factors. a Heatmaps of cytokines and growth factors whose hypoxia-induced expression (1% O2 at 6 h or 48 h, fold-change ≥ 1.5 in two independent experiments) was diminished by BHLHE40-knockdown (KD) in LM cells. The gene expression levels were determined using the Illumina Human HT-12 expression BeadChips. Normalized (quantile normalization) hybridization signals were log2 transformed and standardized by genes across experiment conditions to generate the heatmap. b Heatmaps of a subset of genes list in a whose expression was affected by BHLHE40-KD in LM cells exposed to hypoxia combined with low (1 mM) glucose (1%O2/LG, 4 h). The gene expression levels were determined using the Affymetrix Human Gene 1.0 ST array. c Heatmaps of hypoxia-induced genes whose expression was not significantly affected by BHLHE40-KD in LM cells as determined by the Illumina Human HT-12 expression BeadChips. d Expression of luciferase reporters driven by hypoxia-responsive elements of ITGA6 or LDHA was not affected by BHLHE40 knockout (KO) by CRISPR/Cas9 editing in MDA-MB-231 cells, in the absence or presence of exogenous HIF1A. Luciferase activities were normalized to co-transfected CMV-β-galactosidase and presented as mean ± SD (n = 6). e Expression of genes in control LM empty vector (EV) and LM BHLHE40-KD cells exposed to 1%O2/LG (4 h). mRNA expression levels were determined by qPCR, normalized to RPL13A, and presented as mean ± SD (n = 6). *p <  0.05 (n = 6, 1%O2/LG vs. untreated control), **p <  0.05 (n = 6, KD vs. EV), one-way ANOVA followed by Tukey’s post-hoc tests. f mRNA and protein expression levels of HBEGF and CTGF in primary xenograft tumors, determined by qPCR and immunoblotting, respectively. *p <  0.05 (n = 6, KD vs. EV), Student’s t test. Representative immunoblotting images of three tumors of KD or EV cells are presented