B Cell Translocation Gene 2 (Btg2) Is Regulated by Stat3 Signaling and Inhibits Adipocyte

Supplementary Material

B cell translocation gene 2 (Btg2) is regulated by Stat3 signaling and inhibits adipocyte differentiation

Suji Kim1, Joung-Woo Hong2,* and Kye Won Park1,*

1Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon, 16419, Korea 2Graduate School of East-West Medical Science, Kyung Hee University, Yongin, 17104, Korea

*Corresponding Authors:

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

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

Running Title: BTG2 inhibits adipogenesis. Figure S1. Expression of several known Stat3 target genes in C3H10T1/2 (A-C) and 3T3-L1 (D-

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

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

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).

Figure S3. Expression levels of Stat3 during adipogenic differentiation of C3H10T1/2 (left) and

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

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

Clarkson, R.W., Boland, M.P., Kritikou, E.A., Lee, J.M., Freeman, T.C., Tiffen, P.G., Watson,

C.J. (2006) The genes induced by signal transducer and activators of transcription (STAT)3 and

STAT5 in mammary epithelial cells define the roles of these STATs in mammary development,

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