Fig. 8
The HIF-BHLHE40-HBEGF axis plays a role in promoting cell migration during wound healing. a Monolayer scratch increased protein levels of HIF1A, BHLHE40, and HBEGF in MDA-MB-231 empty vector (EV) cells. Intensive scratch wounds were generated using EMD Millipore’s Cell Comb scratch assay kit and immunoblotting was performed 6 h after cells cultured under normoxia (19% O2) or hypoxia (1% O2). b BHLHE40-knockout (KO) by CRISPR/Cas9 editing reduced the migratory activity of MDA-MB-231 cells, which was restored by the addition of HBEGF peptide into the culture medium. In contrast, a HBEGF-neutralizing antibody reduced the migratory activity of MDA-MB-231-EV cells. Real-time assessment of migratory activity after wound scratch was performed using the IncuCyte ZOOM-ImageLock plate system. *p <  0.05 (n = 6, time points 6–24 h, HBEGF vs. untreated), **p <  0.05 (n = 6, time points 6–24 h, anti-HBEGF vs. untreated), one-way ANOVA followed by Tukey’s post-hoc tests. Representative data from two independent experiments with six replicates are presented. c Images of representative wound fields at 0 and 24 h after wound scratch as described in b. d Conditioned medium or purified exosomes from MDA-MB-231-EV cells (24 h after wound scratch) increased migratory activities of MDA-MB-231 BHLHE40-KO cells, as determined by the transwell migration assays. The migrated cells in six fields were imaged and counted under fluorescent microscopy. The results are presented as: percent migration = mean number of cells migrating through the uncoated transwells × 100/mean number of seeded cells; *p <  0.05 (n = 12, vs. untreated control), one-way ANOVA followed by Tukey’s post-hoc tests