Effects of p130Cas expression on branching morphogenesis in primary mouse mammary gland organoids grown in 3D Matrigel cultures.

<p>(A) Let panel: qRT-PCR expression analysis of p130Cas using total RNA isolated from the 4th inguinal mammary gland of at least three FVB wt mice during different stages of puberty. The amount of p130Cas mRNA was normalized to 18S rRNA. Right panel: Analysis of p130Cas protein expression from wt mice (three mice/stage) at the same stages of puberty as in the left panel. (B) Analysis of p130Cas protein expression from 5 day untreated wt and p130Tg organoids (upper panel) and quantification analysis expressed as the mean of p130Cas/tubulin ± standard deviation (SD) (*p<0.05) (lower panel). (C) Brightfield images of representative wt and p130Tg organoids, after 1, 4 and 5 days of culture in presence of EGF (35 ng/ml) or FGF-2 (35 ng/ml) in the culture medium. Images were captured by using the Axio Observer Z1 microscope. (D) Quantitative analyses of branching morphogenesis after treatment of wt and p130Tg organoids with EGF and FGF-2 at 5 days. The plot represents the mean number of overall branching response in at least three wells from 4 independent experiments. Similar results were obtained with a second p130Cas transgenic line (data not shown). Quantification of the mean number of branched organoids buds was significantly higher in EGF and FGF-2 treated p130Tg organoids compared to wt organoids (*p<0.002).