Terminal Differentiation of Human Breast Cancer through PPARγ Elisabetta Mueller, Pasha Sarraf, Peter Tontonoz, Ronald M. Evans, Katherine J. Martin, Ming Zhang, Christopher Fletcher, Samuel Singer, Bruce M. Spiegelman  Molecular Cell  Volume 1, Issue 3, Pages 465-470 (February 1998) DOI: 10.1016/S1097-2765(00)80047-7

Figure 1 PPARγ Expression in Human Breast Cancer Cell Lines and Tumors (A) Northern blot analysis of breast cell lines and human adipose tissue was performed with 30 μg of total RNA per lane. (B–E) Consecutive sections were stained either with H and E or with a rabbit anti-murine PPARγ antibody at a dilution of 1:1000. (B) and (C) show a histologic section of a breast adenocarcinoma metastatic to lung stained with H and E (B) or PPARγ antibody (C). Note the intense brown nuclear staining of the metastatic adenocarcinoma cells (arrow 1) and the brown nuclear staining of lung pneumocytes (arrow 2). (D) and (E) show a histologic section of normal breast tissue stained with H and E (D) or PPARγ antibody (E). Note the intense brown nuclear staining of the normal breast epithelial cells lining the ducts (arrow 3) as well as the intense brown nuclear staining of the surrounding normal fat cells (arrow 4). Molecular Cell 1998 1, 465-470DOI: (10.1016/S1097-2765(00)80047-7)

Figure 2 Lipid Accumulation in Breast Mammary Epithelial Cells Induced by PPARγ Ligands Staining for neutral lipids was performed with Oil Red O (A and B) and with the fluorescent dye Nile Red, showing neutral lipids in yellow (in C). (A) 21PT cells were treated with 10 μM troglitazone or vehicle (0.1% DMSO; Control) for 7 days. (B) 21MT cells treated with 10 μM troglitazone or vehicle for 15 days. (C) 21PT cells were treated for 5 days with troglitazone (10 μM) or with the M2 compound (10 μM), an inactive metabolite of troglitazone with no affinity for PPARγ. Molecular Cell 1998 1, 465-470DOI: (10.1016/S1097-2765(00)80047-7)

Figure 3 Effects of PPARγ Activation on Gene Expression and Cell Growth (A) Northern blot analysis of RNA from 21PT cells treated for 7 days with 10 μM pioglitazone (Pio), the RXR-selective ligand LG268 (LG268; 100 nM), or with the combination of pioglitazone and LG268 (Pio+LG268) or vehicle alone (0.1% DMSO). 30 μg of total RNA was loaded per lane. (B) Incorporation of thymidine in cultures of exponentially growing 21PT cells, exposed to 10 μM pioglitazone or troglitazone for 3 or 7 days. Cells were then incubated with 2 μCi/ml of 3H-thymidine for a further 24 hr under the same conditions. Error bars represent the standard deviation. (C) Clonogenic growth of 21PT cells treated first with troglitazone or vehicle (0.1% DMSO) for 15 days (see Experimental Procedures) and then replated at 104 cells per 10 cm dish, in the presence or absence of troglitazone (10 μM). Molecular Cell 1998 1, 465-470DOI: (10.1016/S1097-2765(00)80047-7)

Figure 4 MAP Kinase Inhibition Potentiates the Ligand Activation of PPARγ in 21MT Cells (A) Nile Red staining yellow for lipid in 21MT cells treated for 6 days with troglitazone (10 μM), PD98059 (4 μM), or the combination of both. (B) Transcriptional activity of endogenous PPARγ measured by a luciferase reporter gene assay (see Experimental Procedures) in cells treated with vehicle (0.1% DMSO), troglitazone (Tro; 10 μM), PD98059 (PD; 4 μM), and the combination of troglitazone and PD98059 (Tro+PD). Vertical axis represents fold activation. Error bars represent the standard deviation. (C) Northern blot analysis of gene expression in cells treated for 6 days with 10 μM troglitazone, PD98059 (20 μM), the combination of troglitazone and PD98059, or vehicle alone (0.1% DMSO). (D) Western blot analysis of protein extracts from cells cultured with troglitazone (10 μM), with PD98059 (40 μM), with the combination of both, or vehicle (0.1% DMSO) for 4 hr. The antibody against MAP kinase recognizes only the phosphorylated, activated forms of this enzyme. The antibody against PPARγ recognizes both the faster migrating, unphosphorylated form (−) and the slower migrating, phosphorylated form (P). Molecular Cell 1998 1, 465-470DOI: (10.1016/S1097-2765(00)80047-7)