Figure 4

Phenotypic modulation of adipocytes is regulated by Crabp1. (A) Overlap of differentially regulated genes in adipocytes. (B) qRT-PCR of crabp1 transcript in adipocytes isolated from the fourth inguinal mammary gland. (N = nulliparous, P = pregnant, L = lactation, I = involution, R = regressed). Data are presented as average 2^(-ΔCt) ± SEM; n = 3 experiments. (C) Western blot of crabp1 protein in adipocytes as in (B). (D) Immunofluorescence of crabp1 (red) and cytokeratin (green) in mammary glands; DAPI nuclear counterstain (blue). Arrows indicate adipocytes that are crabp1 positive. Scale bar = 100 μm. (E) qRT-PCR of crabp1 transcript in ASC-L treated with shRNA for crabp1 or scrambled control. Data are presented as average 2^(-ΔΔCt) ± SEM; n = 3 experiments. (F) Representative images of Oil Red O staining of ASC-L transduced with shRNA (crabp1 or scrambled control) and exposed to adipocyte differentiation media. (G) Quantification of Oil Red O staining from (F). Data are presented as average absorbance ± SEM. Untreated cell absorbance is set to one; n = 3 experiments. (H) Quantification of glycerol secreted by ASCs normalized to lipoprotein lipase RNA levels. n = 3 experiments. (I) Tumor growth curve and tumor weight (J) of mice injected at eight weeks with 4 T1-12 B cells co-mixed with untreated ASC-L or ASC-L transduced with shRNA (crapb1 or scrambled control) (n = 10 per group). P value indicates significance compared to shScram-ASC-L. Data are means ± SEM. (K) qRT-PCR of VEGF-D in ASC-Ls transduced with shRNA for crabp1 or scrambled control. Data are presented as average 2^(-ΔΔCt) ± SEM; n = 3 experiments. (L) Quantification of tube formation of primary mouse heart endothelial cells treated with media collected from ASC-Ls transduced with shRNA (crabp1 or scrambled control). Data are presented as means ± SEM. n = 3 experiments. ASC-L, lactation-derived adipose stromal cells; crabp1, cellular retinoic acid binding protein-1; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.