Figure 4

ALDH activity is important for mammary stem/progenitor cell functions. (A) Expression of ALDH1A1 and ALDH1A3 in primary human mammary epithelial cells after knockdown (KD) of ALDH1A1 and ALDH1A3, respectively, by using shRNA. (B) Effects of ALDH KD on mammosphere formation. Primary human mammary epithelial cells were infected with shRNA against ALDH1A1 and ALDH1A3, respectively, and cells were plated in suspension at low density and allowed to form mammospheres. The numbers of mammospheres formed were counted after 10 days and compared with nontargeting shRNA (NT). Data are presented as mean number of spheres formed/10,000 plated cells ± SD. (C) Reduction of mammosphere formation after silencing ALDH1A1 by using shRNA was rescued by treatment with 1 μm all-trans retinoic acid (ATRA). (D) Expression of ER in mammospheres from cells infected with shRNA against ALDH1A1 and ALDH1A3, respectively. (E) Quantification of ER expression in mammospheres where ALDH1A1 or ALDH1A3 has been silenced shows that ER expression was significantly reduced in mammospheres from ALDH1A1 KD cells. (F) Effect of ALDH1A1 KD on branching in 3D culture. Cells were infected with shRNA against ALDH1A1 or NT control and embedded in Matrigel. Pictures show representative structures at 13 days of cultivation. (G) Effect of retinoic acid treatment on branching. Matrigel cultures were treated with 1 μm ATRA or DMSO control. Pictures show representative structures at 15 days of cultivation. ALDH1A1 KD was obtained by using a pool of two different shRNAs (see Additional file 7) in all these experiments. Scale bar = 50 μm.