Figure 3

miR-33b inhibits breast cancer cell stemness.

(A) Representative images of the mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. Quantification of primary, secondary and tertiary mammospheres formed by BT-549/ctrl and BT-549/miR-33b cells. (B) Representative images of the mammospheres formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. Quantification of primary, secondary and tertiary mammosphere formation formed by MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (C,D) Tumorsphere assays for BT-549/ctrl and BT-549/miR-33b (C) and MDA-MB-231/ctrl and MDA-MB-231/miR-33b (D) cells were performed by limiting dilution with 1,000 cells to one cell per well of a 96-well ultra-low attachment plate. The numbers of mammospheres were scored at the end of 7 days. This experiment was performed three times, with four wells per cell dilution. (E) FACS analysis of the CD44⁺/CD24⁻ stem cell subpopulation in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/miR-33b cells. (G) Western blot analysis of Bmi-1, Nanog, Oct4, Sox2, HMGA2, SALL4 and Twist1 expression in BT-549/ctrl and BT-549/miR-33b cells as well as in MDA-MB-231/ctrl and MDA-MB-231/ miR-33b cells. The full-length blots were presented in the Supplementary Figure 8. Scale bars, 100 μm. Data represent the mean ± s.d. *: P <0.05, **: P <0.01, ***: P <0.001, N.S.: no significance, N.D.: not detected.