miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation (2016)
Figure 1

Figure 1. Breast CSC enrichment and miRNA expression.

(A) Breast cancer cell lines were sorted using CD44 for luminal MCF-7 cells (left panel) or EpCAM for MDA-MB-231 cells (right panel). Serial mammosphere assays were performed using 4000 cells for each cell fraction, counting spheres after 3 weeks. Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate.

(B) Extreme limiting dilution assay (ELDA) of MCF-7 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in CD44+ cells was 312 cells, compared with 615 cells for both unsorted and CD44- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1

(C) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL, Vimentin and TWIST in MCF-7/CD44+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cell line. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.

(D) miR-10b overexpression affecting stemness and EMT factors. Relative quantification of miR-10b (relative to RNU6B), HOXD10 and RHOC (relative to GAPDH) was performed in MCF-7/CD44+ fractions compared to non-enriched cells. Box plots show the range, median and quartile interval values from 3 independent experiments. A two-tailed Student’s t test was used to compare the two groups (p < 0.05). SEM, standard error of the mean; TLDA, TaqMan Low Density Arrays; and EMT, epithelial-mesenchymal transition.

(E) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA-MB-231/EpCAM+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cells. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.

(F) Extreme Limiting Dilution Assay (ELDA) of MDA-MB-231 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in EpCAM+ cells was 156 cells compared with 625 cells for both unsorted and EpCAM- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1

(G) Nine miRNAs are commonly regulated in both CD44+ and EpCam+ breast cancer cells. Quantitative RT-PCR of miRNAs was performed on sorted MCF-7 and MDA-MB-231 cell populations using TaqMan Low Density Arrays (TLDA). Results were plotted as a heat map comparing CD44+ to CD44- cells (MCF-7) and EpCam+ to EpCam- cells. The scale is shown below the map.