Figure 4

Mammospheres are enriched for Primary Cilia. Mammosphere cultures derived from wild type mammary glands were immunofluorescently stained for PC (A, B) using anti-Arl13B antibody (green). Nuclei were stained with DAPI (blue). Low (A) and high (B) magnification images are shown. The number of mammospheres generated from equal numbers of mammary epithelial cells isolated from control and MMTV-Cre; Ift88^Del glands were determined (C). Mammospheres derived from control, Cre-negative (D) and MMTV-Cre; ROSA26^LacZ (E,F) glands were stained with ß-galactosidase (blue). Mammospheres demonstrated variable levels of staining (E) and staining within mammospheres was chimeric (F; arrows, representative unstained cells). Mammary epithelial cells from Ift88^LoxP/LoxP mice were infected with Ad-GFP or Ad-Cre adenovirus and the number of mammospheres that grew from equal number of cultured cells was determined (G). Deletion of Ift88 was determined in genomic DNA isolated from the cells in mammosphere using PCR to detect Cre and the LoxP, wild type (Wt), and deleted (Del) alleles (H). PC were stained using Arl13B antibody (green) in Ad-GFP (I) and Ad-Cre (J) infected mammospheres. Nuclei were stained with DAPI (Blue). Ad-GFP and Ad-Cre infected mammospheres were dissociated and plated into secondary mammosphere culture and the number of mammospheres generated was counted (K). PC were stained in Ad-GFP (L) and Ad-Cre (M) infected secondary mammopheres using Arl13B antibody (green) and DAPI (blue).