Skip to main content
Fig. 3 | Breast Cancer Research

Fig. 3

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

Fig. 3

BHLHE40 depletion impaired cell migration, invasion, and survival. a BHLHE40-knockdown (KD) by shRNA in LM cells reduced cell migration and invasion, as well as the number of viable cells during suspension culture in comparison to empty vector (EV) control LM EV cells. Migratory and invasive activities were determined using transwells, uncoated or coated with Matrigel, respectively. The results were presented as: percent migration = mean number of cells migrating through the uncoated transwells × 100/mean number of seeded cells; percent invasion = mean number of cells migrating through the Matrigel-coated transwells × 100/mean number of migrating cells through the uncoated transwells. Suspension culture was conducted by seeding cells in medium containing 1% methylcellulose and in dishes coated with PolyHEMA for 15 days. Viable cells were counted under fluorescent microscope. *p <  0.05 (n = 6, KD vs. EV), Student’s t test. b BHLHE40-knockout (KO) by CRISPR/Cas9 editing in MDA-MB-231 cells reduced the number of viable cells after suspension culture and enhanced apoptosis induced by hypoxia combined with glucose depletion (1%O2/GF). Viable cells were determined by Trypan blue exclusion-based cell counting after a 15-day suspension culture. Apoptosis of cells exposed to 1%O2/GF for 6 h was examined by the appearance of apoptotic morphology (as indicated by arrows in the cell images) and Caspase3/7 assays. *p <  0.05 (n = 6, KO vs. EV), one-way ANOVA followed by Tukey’s post-hoc tests. c BHLHE40-KD by shRNA in tamoxifen-resistant subline of MCF7 (TR) reduced the ability of cells to survive 1%O2/GF (6 h) and reduced the number of viable cells after a 15-day suspension culture. Elevated expression of HIF1A and BHLHE40 in TR and fulvestrant-resistant (FR) sublines of MCF7 cells were detected by qPCR. BHLHE40-KD was confirmed by immunoblotting. Apoptosis induced by 1%O2/GF (6 h) was determined by Caspase3/7 assays. The number of viable cells after a 15-day suspension culture was determined by Trypan blue exclusion-based cell counting. **p <  0.05 (n = 6, TR or FR vs. parent MCF7), *p <  0.05 (n = 6, KD vs. EV), one-way ANOVA followed by Tukey’s post-hoc tests

Back to article page