<p>(<b>A</b>) Expression, as measured by qPCR, of <i>Spry2</i> and target genes of FGF signaling, including <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i>, in response to a 24-hour treatment of FGF2 (10 nM) or FGF10 (10 nM). Expression is relative to that of the untreated samples. Values shown are the mean ± standard deviation (SD) of three independent experiments. Statistically significant differences of p<0.05 (t test) were observed between expression of untreated and treated samples for all genes except for <i>Etv5</i> in response to FGF2 and FGF10 treatment. (<b>B</b>) Schematic diagram depicting the experimental procedure in sample preparation, treatment, and analysis. Mammary organoids were prepared from <i>Spry2</i><sup>+/+</sup> and <i>Spry2</i><sup>fl/fl</sup> mice and were infected with adenovirus-Cre-GFP, which generated control (<i>Spry2</i><sup>+/+</sup>) and mutant (<i>Spry2</i><sup>Δ/Δ</sup>) organoids, respectively. Transduced cells were then purified by FACS based on their expression of GFP before they were subjected to analyses on gene expression and epithelial morphogenesis in the presence or absence of FGF2 or FGF10. (<b>C–D</b>) Expression, as measured by qPCR, of <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i> in control and mutant MECs in response to 24-hour treatment of FGF2 (200 ng/ml, <b>C</b>) or FGF10 (200 ng/ml, <b>D</b>). Expression is relative to that of the control samples. Statistically significant differences of p<0.05 (t test) were observed between expression of control and mutant samples for all genes except for <i>Etv5</i> in response to FGF2 treatment and <i>Etv4</i> in response to FGF10 treatment. (<b>E–I</b>) in vitro branching assay in which control (<b>E</b>, <b>F</b>) and mutant organoids (<b>G</b>, <b>H</b>) were subjected to cultures in basal medium with (<b>F</b>, <b>H</b>) or without FGF2 (<b>E</b>, <b>G</b>). When stimulated by FGF2 at progressively higher concentrations from 0.025 nM to 0.5 nM, a progressively higher percentage of organoids underwent branching. At 1.0 nM and 2.5 nM, FGF2 did not stimulate a higher percentage of branched organoids to form. In addition to their differences in branching kinetics, <i>Spry2</i><sup>Δ/Δ</sup> organoids overall formed larger branched structures than control organoids. Scale bars: 100 μm. (<b>I</b>) Quantitative comparisons of control and mutant MECs in their ability to undergo epithelial branching in vitro. Data were from experiments repeated three times or more. At least 100–150 organoids were examined for each treatment conditions. Values shown are the mean ± SD for each data point: *P<0.0005, unpaired, two-tailed Student’s <i>t</i> tests.</p
