Fig. 7
BHLHE40 depletion reduced phosphorylation of epidermal growth factor receptor (EGFR), while it increased Caspase 9 cleavage, in cells exposed to glucose depletion and hypoxia (1%O2/GF). a BHLHE40-knockout (KO) by CRISPR/Cas9 editing in MDA-MB-231 and BHLHE40-knockdown (KD) by shRNA in tamoxifen resistant (TR) cells diminished HBEGF induction by 1%O2/GF (6 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/GF vs. control), **p <  0.05 (n = 6, KO vs. EV), one-way ANOVA followed by Tukey’s post-hoc tests. b BHLHE40 depletion reduced EGFR activation, as indicated by reduced phosphorylation of EGFR and its downstream targets (ERK and AKT), while increasing apoptosis, as indicated by detection of cleaved caspase 9. Data from three independent immunoblotting analyses are presented. c HBEGF peptide (10 μg/ml) reduced apoptosis induced by 1%O2/GF (6 h) in MDA-MB-231 BHLHE40-KO cells. Apoptosis was determined by Caspase 3/7 assays. *p <  0.05 (n = 6, 1%O2/GF vs. control), **p <  0.05 (n = 6, HBEGF vs. untreated with HBEGF), one-way ANOVA followed by Tukey’s post-hoc tests