Fig 1: The expression and the activity of breast cancer stem cell (BCSC) markers were decreased in NEDD4-deleted MDA-MB-231. (A) The expression of BCSC markers including ALDH1A1 and CD44 was confirmed by western blot in siCONT- and siNEDD4-treated MDA-MB-231. ß-actin was used as a loading control. (B) The activity of ALDH1A1 was confirmed by the ALDEFLUOR kit system in siCONT/NEDD4-treated MDA-MB-231 cells. The representative images are shown. The average value is shown in the graph (n = 3). (C) siCONT/NEDD4-treated MDA-MB-231 cells were induced to mammosphere formation for 2 weeks. The representative images are shown (n = 3). (D) The lysates harvested from the mammosphere formation were subjected to western blot using the indicated antibodies. ß-actin was used as a loading control. The representative images are shown. The average value is shown in the graph (n = 3) (**P < 0.01 and ***P < 0.001).
Fig 2: Low expression of RICH1 is associated with the enhancement of CSC properties in breast cancer.A RICH1 expression in primary bulk tumor samples and mammospheres provided by GSE7515. B RICH1 mRNA expression levels in BCSC-like subpopulations (CD44+CD24-, ALDHhi and mammospheres) and non-BCSC-like subpopulations (non-CD44+CD24-, ALDHlow and adherent monolayer). C Correlation analysis between RICH1 and ALDH1A1 intensities in 30 breast cancer samples. D ALDH1A1 expression levels in breast cancer samples with different RICH1 expression. E Representative IHC images of RICH1 and ALDH1A1 of 2 breast cancer patients, PATIENT#1 (case number of #1525760) and PATIENT#2 (case number of #1392301). Among them, PATIENT#1 represented an example with high expression of RICH1 and low expression of ALDH1A1, while PATIENT#2 represented another example with low expression of RICH1 and high expression of ALDH1A1. Data represent mean ± SD. Statistical significance was determined by two-tailed unpaired t-test (A, B), pearson’s r (C) or chi-squared test (D). Abbreviation: CSC cancer stem cell, BCSC breast cancer stem cell, ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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