(A) Mammary gland thin sections from the Col1a1-GFP reporter mouse immunofluorescence stained for GFP⁺ collagen 1–expressing cells (red), fibroblast PDGFRα (green), and myoepithelial cell calponin (purple), show colocalization of GFP and PDGFRα (yellow), but not calponin. Nuclei stained with DAPI (blue). White scale bar: 50 μm. Yellow scale bar: 10 μm. Arrows show PDGFRα⁺GFP⁺calponin^– cells. (B) Flow gate for isolating murine mammary PDGFRα⁺ cells. (C) RT-qPCR on FACS-isolated PDGFRα⁺ cells for fibroblast markers (Col1a1, Col3a1, Col5a1, and PDGFRα) and markers of potential contaminating cell populations: Cd45 (lymphocyte common antigen), F4/80 (mature macrophage), Cd31 (endothelial cell), Adipoq (adipocyte), and Ecad (epithelial cell). Sorted PDGFRα-negative cells were used as positive controls for Cd45, F4/80, Cd31, Adipoq, and mammary epithelial EpH4 cells were used as the Ecad-positive control. n = 3–4 per group. (D) RNA-Seq heatmap illustrates 870 significantly differentially expressed genes (>1.3-fold changes) between fibroblasts isolated from nulliparous and involution day 6 (InvD6) mammary glands. Red: relative high expression. Blue: relative low expression. (E) Pathway analysis of RNA-Seq data set showing extracellular matrix (ECM) regulation–related pathways that are upregulated in InvD6-fibroblasts. (F) PDGFRα⁺ fibroblasts reported as number per gram of mammary tissue observed in nulliparous and 4-, 6-, and 8-day postweaning mice; n = 3–10 per group. (G) Fibrillar collagens and Lox gene expression analysis by RT-qPCR in sorted PDGFRα⁺ mammary fibroblasts from nulliparous and 4-, 6-, and 8-day postweaning mice; n = 3–10 per group. (H) Mmp2, Mmp3, and Tgfb1 gene expression analysis by RT-qPCR in nulliparous and InvD6 fibroblasts, n = 6–9 per group. (I) Immunofluorescence staining shows non-colocalization of α-smooth muscle actin (red) and PDGFRα (green) in mammary glands of nulliparous and InvD6 mice. Boxes show images at high magnification. Arrow: PDGFRα⁺ cells. Arrowhead: αSMA⁺ cells. White scale bar: 50 μm. Yellow scale bar: 10 μm. (J) Pathway analysis of RNA-Seq data described in D showing immune regulation–related pathways that are upregulated in InvD6 fibroblasts. (K) RNA-Seq data show chemokine expression pattern of fibroblasts isolated from nulliparous mammary glands. (L) RT-qPCR gene expression for Cxcl12 in sorted PDGFRα⁺ fibroblasts from nulliparous and InvD6 mammary glands, n = 3–5 per group. Gene expression data are normalized to Actb and are from 2 to 6 independent studies. RNA-Seq data are archived at the NCBI Gene Expression Omnibus (GEO GSE94761). *P < 0.5, **P < 0.01, ***P < 0.001, ^#P < 0.0001 by unpaired 2-tailed t test. Data represent mean ± SEM.