Fig. 1: Loss of Spry1, Spry2, and Spry4 in mammary stromal fibroblasts causes accelerated epithelial branching.

A Schematic diagram depicting the experimental procedures during sample preparation, in vitro culture, and treatment methods. Mammary organoids and stromal fibroblasts were prepared from wild type (WT) and Spry1^fl/fl; Spry2^fl/fl; Spry4^fl/fl mice. Fibroblasts with and without Spry1, Spry2, and Spry4 function were acquired by infection with Ad-Cre-GFP and FACS-mediated selection of GFP-positive infected cells. Wild type organoids were co-cultured with either control fibroblasts or Spry1,2,4-KO fibroblasts, both of which embedded in Matrigel. Abbreviation: LN, lymph node. B–D Wildtype organoids co-cultured with control (B) and Spry1,2,4-KO fibroblasts (C). D Quantification of the number of organoid branches co-cultured with fibroblasts (Fibs). E–G Wildtype organoids cultured using conditioned medium (CM) from control (E) or Spry1,2,4-TKO fibroblasts (F). G Quantification of the number of organoid branches cultured using medium from control or Spry1,2,4-KO fibroblasts. Data were from three independent experiments and were presented as mean ± SD. **P < 0.01; ***P < 0.001. Scale bars: 50 μm. H–O Branching trees of the #4 mammary glands, as revealed by Carmine Red staining of control (H–J) and mutant Spry-TKO (K–M) whole-mount glands, at the 5-wk, 7-wk, and 12-wk stages. Female mice in each comparison group were from the same litter and were checked to ensure that they were in the same estrous state before sacrifice. Arrows indicate the extent of ductal penetration in the fat pad. The dotted white line illustrates the epithelial invasion front. Insets show closer view of the area in a square with black dotted line. N, O Quantitative comparisons of ductal penetration and branching points per millimeter between control and mutant glands. Plots show mean ± SD (n \(\ge\)3/genotype); ns, not significant; *P < 0.05; ***P < 0.001; Scale bars: 5 mm.