1. Small glutamine-rich tetratricopeptide repeat–containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome 
Miriam S. Butler, Ph.D., Xing Yang, M.D., Ph.D., Carmela Ricciardelli, Ph.D., Xiaoyan Liang, M.D., Robert J. Norman, M.D., Wayne D. Tilley, Ph.D., Theresa E. Hickey, Ph.D. 
Fertility and Sterility 
Volume 99, Issue 7, Pages e1 (June 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
(A) Western blot analysis of small glutamine-rich tetratricopeptide repeat–containing protein alpha (SGTA) produces a single band at ∼30 kDa in protein lysates derived from an ovarian granulosa tumor cell line (KGN) and a prostate cancer cell line (C42B). 5α-Dihydrotestosterone (DHT) treatment of the cells for 24 hours increased immunoreactive androgen receptor (AR) protein levels but did not alter immunoreactive SGTA protein levels. α-Tubulin was used as a loading control. Western blot is a representative example of two experiments. (B) SGTA mediates AR subcellular localization in KGN cells. Under control conditions (cells transfected for 48 hours with nonspecific small interfering (si) RNA [siNS]), SGTA is cytoplasmic and AR is predominantly cytoplasmic with some AR nuclear localization observed. In cells transfected with SGTA-specific siRNA (siSGTA), SGTA levels are visually reduced and AR is predominantly localized to the nucleus of the cells, even in the absence of DHT. Magnification at ×200. (C) Western blot confirmation of SGTA knockdown. SGTA level is reduced in siSGTA-transfected KGN cells but not in siNS or no-siRNA control samples. (D) siSGTA- and siNS-transfected cells from duplicate samples in two independent experiments were scored as exhibiting predominantly nuclear AR localization by manual counting. Data are presented as mean percentage of total cells counted for each treatment (0–10 nmol/L) of DHT. ∗ P<.05 compared to siNS.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Immunohistochemical staining of small glutamine-rich tetratricopeptide repeat–containing protein alpha (SGTA) in normal ovarian structures. Ovarian (A) surface epithelial cells (ose) and (B) granulosa cells (g) of primordial follicles, (C) primary follicles, and (D) secondary follicles were consistently positive for cytoplasmic SGTA expression. (E, F) Antral follicles demonstrate intense SGTA expression in the granulosa and theca (t) compartments. Inset: negative control (minus primary antibody). Surrounding stromal (s) cells at all follicle stages had no or very low levels of SGTA. (G) Positive staining within the cytoplasm of Leydig cells (L) of the human testes. (H) Positive control (human prostate cancer tissue) possessed SGTA immunoreactivity in the cytoplasm of the prostate tumor epithelial (e) cells and was absent in the stroma, in agreement with earlier analysis (13), Scale bar = 100 μm.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Small glutamine-rich tetratricopeptide repeat–containing protein alpha (SGTA) mRNA levels in human cumulus cells. Total RNA was extracted from cumulus cells (n = 19) and reverse transcribed to obtain cDNA, which was then subjected to real-time polymerase chain reaction. Expression of SGTA was normalized to L32 and GUSB. Expression levels are displayed (A) per individual samples and (B) as a box-plot to demonstrate the mean level and distribution for non–polycystic ovary syndrome (non-PCOS) and PCOS groups. Compared with cumulus cells from non-PCOS patients, SGTA expression was not significantly different in PCOS patients (P=.724).
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

5. Supplemental Figure 1
To confirm specific staining, the SGTA antibody was incubated with peptide for 1 hour before addition to tissue and overnight incubation at 4°C following the standard immunohistochemical protocol, which resulted in the absence of small glutamine-rich tetratricopeptide repeat–containing protein alpha (SGTA) staining in the granulosa cells (G) and oocyte (Oo).
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions