Differentiation of mammary gland epithelial cells in control (Ctr) and Miz1ΔPOZ tissue and in HC11 cells with reduced levels of Miz1.

(A) Gene expression of the milk proteins α-casein, ß-casein and whey acidic protein (Wap), measured by quantitative RT-PCR, in control and Miz1ΔPOZ mammary gland tissue (ΔPOZ). (B) Immunoblot analysis for ß-casein from mice with the indicated phenotype. Each lane represents an individual animal. (C) Immunostaining with an antibody against milk proteins in the acini from control and Miz1ΔPOZ mammary glands. (D) Sudan III staining was performed on cryosections from lactating mammary glands of Ctr and MizΔPOZ mice (n = 4 per genotype). Data from A to D was obtained from lactation day 6 samples. (E) Time course of growth and differentiation as performed in the experiments with HC11 cells. EGF: epidermal growth factor; DIP: differentiation media containing dexamethasone, insulin and prolactin. Time points indicated correlate to the time points in (G) and (H). (F) HC11 cells were stably transfected with scrambled short hairpin (sh) RNA (shscr), a Miz1 shRNA and a vector expressing Miz1 as a positive control. The film was exposed 1 and 5 minutes, respectively. (G) PCR and (H) Western blots revealed a down-regulation of ß-casein when Miz1 concentration is decreased. Numbers indicated in (B) and (H) are fold changes of band intensities obtained by densitometry (see Materials and Methods). Scale Bar in C: 50 µm; D: 100 µm.