<p>Supplementary Material</p><p>B cell translocation gene 2 (Btg2) is regulated by Stat3 signaling and inhibits adipocyte differentiation </p><p>Suji Kim1, Joung-Woo Hong2,* and Kye Won Park1,*</p><p>1Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon, 16419, Korea 2Graduate School of East-West Medical Science, Kyung Hee University, Yongin, 17104, Korea</p><p>*Corresponding Authors:</p><p>Joung-Woo Hong, Ph.D Graduate School of East-West Medical Science Kyung Hee University Yongin, 17194, Korea E-mail: [email protected] Phone: +82-31-201-3853 Fax: +82-31-204-8119</p><p>Kye Won Park, Ph.D Dept. of Food Science and Biotechnology Sungkyunkwan University Suwon, 16419, Korea Email: [email protected] Phone: +82-31-290-7804 Fax: +82-31-290-7882</p><p>Running Title: BTG2 inhibits adipogenesis. Figure S1. Expression of several known Stat3 target genes in C3H10T1/2 (A-C) and 3T3-L1 (D-</p><p>F) cells in the presence and absence of WP1066 was assessed by real-time PCR. Btg2 expression was increased by a STAT3 inhibitor, WP1066, in both types of cells (Fig. 3A-C). STAT3 is known to regulate transcription of several target genes such as B-Cell CLL/Lymphoma 2 (Bcl2), krüppel-like factor 5 (Klf5) and p53 (Clarkson, R.W. et al, 2006). Transcription of a representative Stat3-upregulated gene Bcl2 (A and D) was significantly repressed upon WP1066 treatment in both types of cells. In contrast, expression of Stat3-downregulated genes Klf5 (B and</p><p>E) and p53 (C and F) was activated by WP1066. The expression measurements were normalized by the transcript level of a 36b4 gene encoding acidic ribosomal phosphoprotein P0. Data shown represent the means ± SD. Statistical significance was determined relative to a control by</p><p>Student’s t-test (* P<0.05). Figure S2. Expression of Stat3 target genes in C3H10T1/2 cells transfected with the control scramble and Stat3-specific siRNAs was measured by real-time PCR. siRNA-mediated knockdown of Stat3 significantly repressed transcription of Stat3-upregulated genes C/ebpδ (A) and Bcl2 (B). In contrast, silencing Stat3 with siRNAs activated expression of Stat3- downregulated genes Klf5 (C) and p53 (D). The measurements were normalized by the transcript level of a 36b4 gene. Data shown represent the means ± SD. Statistical significance was determined relative to a control by Student’s t-test (* P<0.05).</p><p>Figure S3. Expression levels of Stat3 during adipogenic differentiation of C3H10T1/2 (left) and</p><p>3T3-L1 (right) cells are shown. Stat3 transcript levels were assessed by real-time PCR at various time points over 6 days after treatment with a defined adipogenic cocktail including dexamethasone, isobutylmethylxanthine, and insulin (DMI). Transient induction of Stat3 mRNA expression was observed 4 days after adipogenic induction. All expression levels were normalized to that of 36b4. Data are expressed as means ± SD. Statistical significance was determined relative to control samples by Student’s t-test (* P<0.05; ** P<0.005). Figure S4. Specific knockdown of Btg2 SiRNA. siRNA-mediated knockdown of Btg2 in</p><p>C3H10T1/2 cells does not influence the expression of Btg1, a most closely related BTG family member. Knockdown of Btg2 expression also does not affect Stat3 expression. All expression levels were normalized to that of 36b4. Values are expressed as means ± SDs of representative data from three independent experiments. Statistical significance was determined relative to control cells by Student’s t-test (* P<0.05; ** P<0.005). Reference</p><p>Clarkson, R.W., Boland, M.P., Kritikou, E.A., Lee, J.M., Freeman, T.C., Tiffen, P.G., Watson,</p><p>C.J. (2006) The genes induced by signal transducer and activators of transcription (STAT)3 and</p><p>STAT5 in mammary epithelial cells define the roles of these STATs in mammary development,</p><p>Mol. Endocrinol. 30, 675-685.</p>