Fig. 2

Rac1, but not integrin-linked kinase (ILK), is involved in the formation of mammary organoids. a Primary mammary epithelial cells (MECs) were isolated from ILK^fxfx;CreESR mice and cultured as single cells in organoid media in the absence or presence of 4-hydroxytamoxifen (4-OHT). Gene expression levels were quantified using qRT-PCR. b ILK gene deletion has no significant effect on solid or hollow organoid formation after 10 days of culture (n = 2). Error bars = SEM (Student’s t test for paired samples). Representative images of organoids are shown to the right. c Primary MECs were isolated from Rac1^fxfx;CreESR mice and cultured as single cells in organoid media in the absence or presence of 4-OHT. Gene expression levels were quantified using qRT-PCR. d Rac1 gene deletion decreases solid but not hollow organoid formation after 10 days of culture (n = 2). Error bars = SEM (Student’s t test for paired samples). Representative images of organoids are shown to the right. e EHT1864 treatment reduces Rac1 activity in MECs. Primary MECs from ICR mice were cultured with 0, 10, or 20 nM EHT1864, and Rac1 activity levels were measured. f Inhibition of Rac1 activity using EHT1864 reduces solid organoid formation (n = 3). Error bars = SEM (Student’s t test for paired samples). Representative images of organoids are shown to the right. g Rac1 rescues the impaired solid organoid formation caused by the β1-integrin knock-down, with representative images of cultures for control, sh-β1, and sh-β1 + Rac1. Scale bar = 500 μm. h Quantification of solid and hollow organoids demonstrates that Rac1 expression can rescue β1-integrin loss-of-function phenotype for solid but not hollow organoids (n = 4). Error bars = SEM (statistical significance determined by one-way analysis * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 of variance)