Figure 2.

Phenotypic modulation of adipocytes during mammary gland development. (A) Quantification of percent area per tissue occupied by adipose stroma (AS) or parenchyma (P) using Spot software (n = 5 glands per stage). P values indicate significance compared to nulliparous. (B) Representative H & E-stained sections of mammary gland adipocytes isolated from non-diseased mice. Arrows indicate lipolytic adipocytes. (C) Average diameter (μm) of adipocytes. n = 3 glands per stage. P value indicates significance compared to nulliparous. (D) Quantitative RT-PCR of FABP4 and HSL expression in adipose stromal cells. Data are presented as average 2^(-ΔCt) ± SEM; n = 3 experiments. (E) Representative immunofluorescence images of adipose stromal cells isolated from lactating or regressed mammary gland and NIH3T3 cells (negative control), 3T3L1 cells (positive control) with inset (no primary) for FABP4 (red) and counterstained for nuclei with DAPI (blue) (n = 3 per group). (F) Representative images of Oil Red O staining and quantification (G) of adipose stromal cells. Line indicates untreated cells whose absorbance is set to one; n = 3 experiments. (H) Quantification of glycerol secreted by adipose stromal cells normalized to lipoprotein lipase RNA levels. n = 3 experiments. (I) Quantitative RT-PCR of Klf2, PPARγ, FABP4 in adipose stromal cells treated with adipocyte differentiation media. Data are presented as fold change in comparison to undifferentiated cells. Horizontal black line indicates undifferentiated cells set to one. Bars above the black line indicate an increase in expression compared to undifferentiated cells. Bars below the black line indicate a reduction in expression compared to undifferentiated cells. Data are presented as average 2^(-ΔΔCt) ± SEM. n = 3 experiments. (J) Gene set enrichment analysis of transcriptional signature of adipose stromal cells from lactating mice. Scale bar = 100 μm. All data are means ± SEM. DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.