Skip to main content
Fig. 5 | Breast Cancer Research

Fig. 5

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

Fig. 5

Exosomic secretion of HBEGF was reduced by BHLHE40 depletion in MDA-MB231 or TR cells. a BHLHE40-knockout (KO) by CRISPR/Cas9 editing in MDA-MB-231 cells reduced the total number of exosomes and the amount of HBEGF protein in exosomes in comparison with empty vector (EV) control cells. b BHLHE40-knockdown (KD) by shRNA in tamoxifen-resistant (TR) cells reduced the total number of exosomes and the amount of HBEGF protein in exosomes in comparison with TR EV cells. Exosomes were purified from conditioned medium of 5 × 106 cells cultured in medium supplemented with exosome-free serum, either under normal culture condition or exposed to 1% O2/low glucose (LG) for 6 h. Exosomes were purified using the ExoQuick-TC solution and quantified under a fluorescent microscope after being labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) using the Exo-Glow labeling kit, which is designed to exclude background particles. Exosome number in the bar graph is presented as mean number of exosomes per field ± SD (total of nine fields from three independent experiments were examined). The presence of HBEGF and exosome markers in purified exosomes (3 μg protein/lane) or whole cell extracts (WCE; 30 μg protein/lane) were detected by immunoblotting. **p <  0.05 (n = 9, 1%O2/LG vs. control), *p <  0.05 (n = 9, KO or KD vs. EV), one-way ANOVA followed by Tukey’s post-hoc tests

Back to article page