1. Smad7 is a transforming growth factor-beta–inducible mediator of apoptosis in granulosa cells 
Marisol Quezada, Ph.D., Jikui Wang, Ph.D., Valerie Hoang, M.S., Elizabeth A. McGee, M.D. 
Fertility and Sterility 
Volume 97, Issue 6, Pages e6 (June 2012)
DOI: /j.fertnstert
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Localization and regulation of Smad6 and Smad7 in the ovary. (A) Smad6 is highly expressed in the cytoplasm of the oocytes of growing follicles. (B) Smad7 is expressed in the granulosa cells and oocytes of growing follicles and in corpora lutea. (C) Negative control is absence of primary antibody. PAF = preantral follicle; AF = antral follicle; CL = corpus luteum. (D) Effect of TGF-β (1 ng/mL) on Smad6 and Smad7 mRNA from primary granulosa cells cultured. Mean ± SEM; aP<.05.
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3. Figure 2
Transforming growth factor-β responsiveness of the Smad7 promoter. (A) SIGC cells were transfected with a 4.3-kb mouse Smad7 promoter reporter construct and treated as indicated. Transforming growth factor-β treatment increased luciferase activity, but activin and stimulators of PKA did not. (B) Putative SBE sites of Smad7 promoter (black circles). The progressive 5′ deletion fragments are represented by grey bars. Mutated SBE is depicted by the black rectangle. Mean ± SEM; a ≠ b,b ≠ cP<.05.
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4. Figure 3
Neither Smad2 nor Smad3 are specifically required for up-regulation of Smad7. (A) SIGC cells transfected with Smad7 promoter were incubated for 24 hours with TGF-β (1 ng/mL) in and SB μM or vehicle control. (B) Smad7 mRNA expression in primary granulosa cells treated as above. (C) SIGC cells transfected with the Smad7 promoter and incubated for 24 hours with or without TGF-β (1 ng/mL) in the presence of the specific inhibitor of Smad3 phosphorylation (SIS3, 10 μM) or vehicle control. Luciferase activity was normalized against activity of a cotransfected renilla vector. Bars represent mean ± SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells from wild-type or from Smad3 knockout mice (hatched bars) or from wild-type primary cells transfected with siRNA for Smad2 (grey bars) incubated 24 hours with the treatments before mRNA analysis as above. Mean ± SEM; a ≠ bP<.05.
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5. Figure 4
Smad7 overexpression induces apoptosis, whereas TGF-β–induced apoptosis requires Smad7 expression. Primary granulosa cells transfected with full-length Smad7 cDNA (S7FL) or EV and treated with TGF-β (1 ng/mL). (A) Cell viability determined by tetrazolium salt assay. (B) Programmed cell death was assessed with TUNEL. At both 6 and 24 hours S7FL cells were overwhelmingly more apoptotic than EV cells. (C) Assay (TUNEL) of primary granulosa cells treated with TGF-β (black bars) or vehicle (white bar). Cells were transfected with siRNA Smad7 or control siRNA before TGF-β treatment. Mean ± SEM; a ≠ b,c ≠ dP< .05.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Regulation of Smad7 in granulosa cells. (A) Transforming growth factor-β dose–response effect on Smad7 mRNA expression in mouse granulosa cell primary culture. (B) Transforming growth factor-β dose–response effect on Smad7 promoter activity in SIGC cells transfected with the 4.3-kb mouse Smad7 promoter reporter construct. Luciferase activity was normalized against activity of a cotransfected renilla vector. In both, Smad7 expression was maximal at a dose of 10 ng/mL. Bars represent mean ± SEM from three different experiments. (C) Smad7 mRNA expression in primary granulosa cells treated with activin A (200 ng/mL). Activin treatment did not increase Smad7 mRNA expression levels. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the ΔΔCT method. Results represent mean ± SEM from three different experiments. (D) Smad7 mRNA expression in primary granulosa cells treated with FSH (1 mIU/mL) and/or TGF-β. Treatment with FSH did not modify either basal or TGF-β–stimulated Smad7 mRNA expression. Data were normalized with the housekeeping gene TBP and expressed as fold change of the control using the ΔΔCT method. Results represent mean ± SEM from three different experiments.
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7. Supplemental Figure 2
Additional data confirming inhibitor and siRNA effects on gene expression. (A) Expression of PAI-1 mRNA in primary granulosa cells treated with SIS3 (10 μM). SIS3 preincubation blocked TGF-β–stimulated PAI-1 increase, confirming that SIS3 is able to function as an inhibitor in granulosa cells. (B) Primary granulosa cells cultured on coverslips were transfected with siRNA-Smad2. (C) Primary granulosa cells were also transfected with siRNA-Smad7. The intensity of Smad2 and Smad7 protein expression per cell decreased markedly compared with the negative control. Bars represent mean ± SEM from three different experiments.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

8. Supplemental Figure 3
Smad7 promoter. Schematic map of mouse Smad7 promoter region with putative cis-regulatory elements from (−500 to +280 bp), which contains three palindromic SBE sites. Androgen receptor (AR), nuclear factor of activated T cells (NFAT), specificity protein 1 (SP1), specificity protein 3 (SP3), peroxisome proliferator-activated receptor-α (PPARα), and activator protein 1 (AP1) were additional transcription factor–binding sites that were adjacent to the SBE sites. The inset sequence spans from −332 to −85 bp and shows the most proximal SBE and its 3-bp spacing from the E-box and overlapping AP-1 and c-Myc sites.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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9. Supplemental Figure 4
Smad7 overexpression reduces TGF-β–stimulated PAI-1 expression. (A) Primary granulosa cells were transfected with full-length Smad7 (S7FL), truncated Smad7 (S7T), Smad7, EV, or NT and subject to 24 hours of TGF-β treatment (1 ng/mL). Treatment with TGF-β increased the expression of PAI-1 mRNA for NT, EV, and S7T groups. However, the cells transfected with full-length Smad7 exhibited a diminished TGF-β–stimulated PAI-1 expression, demonstrating the functionality of this construct in our system. (B) Immunocytochemical analysis for Smad7 in mouse granulosa primary culture after Smad7 full-length transient transfection (S7FL) or transfection with empty vector (EV). The S7FL cells have increased cellular staining for Smad7 relative to EV cells, where staining is seen only at a low level, predominantly in the nucleus. Right panels are of blue DAPI staining for the nucleus (original magnification, ×200). (C) Smad7 protein expression exceeded 1.5-fold in cells transfected with full-length Smad7 (S7FL) compared with the negative control, cells transfected with EV. Bars represent mean Smad7 intensity of fluorescence per cell.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions