FIGURE 4.

ELEANORS activate CD44 gene expression for breast cancer stemness. (A) Higher tumorsphere‐forming ability is shown in HCC1428‐long‐term estrogen deprivation (LTED) cells than in HCC1428 cells. Images of tumorspheres were captured (left) and quantified (right). (B) FACS analysis of stem cell surface markers, CD44⁺/CD24^−/low, in HCC1428‐LTED cells with ELEANOR2 knockdown (left) and quantification (right). (C, D) Quantitative RT‐PCR (qRT‐PCR) analyses of ESR1 mRNA, ELEANORS, and CD44 mRNA. CD44 expression was increased in HCC1428‐LTED cells, which overexpress ELEANORS, compared to HCC1428 cells (C). CD44 expression was reduced with ELEANOR knockdown (D, right columns). (E) Immunoblot analysis of CD44 (top) and its quantification (bottom). (F) FACS analysis of the CD44+ cell population (left) and its quantification (right) in HCC1428‐LTED cells. The number of CD44+ cells was reduced by ELEANOR knockdown. The most frequent value in the analysis was set at 100. (G) Quantitative RT‐PCR analyses of ESR1 mRNA, ELEANORS, and CD44 mRNA. CD44 expression was not reduced by ESR1 mRNA knockdown. (H) Immunoblot analysis of estrogen receptor‐α (ERα) in HCC1428‐LTED cells treated with the indicated siRNAs and locked nucleic acids (LNAs). (E, H) GAPDH served as a loading control. FACS data (B, F) are representative of three independent experiments. (A–G) Values are mean ± SE. p values were determined by independent sample t‐tests: *p < 0.05, **p < 0.01, ***p < 0.001