Characterization of a mechanism to inhibit ovarian follicle activation Sarah J. Barilovits, Ph.D., Kimberly J. Newsom, Ph.D., Justin S. Bickford, Ph.D., Dawn E. Beachy, B.F.A., Alice Rhoton-Vlasak, M.D., Harry S. Nick, Ph.D.  Fertility and Sterility  Volume 101, Issue 5, Pages 1450-1457.e4 (May 2014) DOI: 10.1016/j.fertnstert.2014.01.025 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Demonstration of Foxo3 induction by nutrient deprivation. (A) Foxo3 mRNA was analyzed by qRT-PCR in SKOV3 cells treated with HisOH or 2-DG for indicated time periods (n≥3). *P≤.05. (B) A representative immunoblot shows Foxo3 in SKOV3 cells treated with HisOH or 2-DG for indicated time periods. (C) Foxo3 mRNA was analyzed by qRT-PCR in IOSE80PC, JH514 and OVCAR3 cells treated with 2-DG for 24 hours (n = 2). Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Chromatin immunoprecipitation (ChIP) analysis of the Foxo3 gene and Foxo3 localization and induction in mouse ovaries. (A) Top: The genomic structure of the Foxo3 gene is shown, with the four exons (E1–4) indicated. The ATF4 consensus binding sites (sites 1–4) are indicated by location in the gene and sequence. Bottom: Pol II and ATF4 associated with the Foxo3 promoter and site 2 were analyzed by ChIP. The SKOV3 cells were treated with 2-DG for 24 hours before crosslinking for ChIP analysis. Association with the Foxo3 gene was analyzed by qPCR (n ≥ 2). (B) A representative immunohistochemical analysis of Foxo3 protein shows localization in mouse ovaries. Postnatal day (PD) 1 and PD17 mouse ovaries from B6129 mice were fixed and stained for Foxo3 expression and localization. (C) Foxo3 mRNA was analyzed by qRT-PCR in mouse ovarian organ culture. Ovaries from C57BL/6 mice (n = 2) were treated with 2-DG for 24 hours. Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Analysis of gene and protein expression in 2-DG-treated mouse organs. Mice were treated as described in Materials and Methods. (A–D) Gene expression was analyzed by qRT-PCR, with four to five animals per treatment group. *P≤.05. (A) Foxo3, GRP78, CHOP, ATF4, and ASNS mRNA were analyzed in kidneys. (B) Foxo3, GRP78, CHOP, ATF4, and ASNS mRNA were analyzed in brains. (C) Foxo3 mRNA expression was analyzed in ovaries. (D) GRP78, CHOP, ATF4, ASNS, and c-Myc mRNA expression was analyzed in ovaries. (E) A representative immunohistochemical analysis shows ATF4 expression in PBS-treated and 100 mg/kg 2-DG-treated mouse ovaries. The bottom panels represent a higher magnification of the boxed areas in the respective top panels. Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of 2-DG on ovarian folliculogenesis. (A) Representative hematoxylin-stained sections illustrate type 3 primordial follicles (arrowheads) in PBS-treated (left) and 100 mg/kg 2-DG-treated (right) mouse ovaries. (B) Activated follicle types in PBS-treated and 2-DG-treated ovaries were counted and classified as described in Materials and Methods, and each type is shown as a percentage of the total activated follicles (n ≥ 3). *P≤.05. Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Foxo3 mRNA analyzed by qRT-PCR in SKOV3 cells treated with 2-DG or mannoheptulose (MH) for 8 hours (n = 2). Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Chromatin immunoprecipitation (ChIP) analysis of putative ATF4 binding sites. Top: The genomic structure of the Foxo3 gene is shown, with the four exons (E1–4) indicated. The ATF4 consensus binding sites (sites 1–4) are indicated by location in the gene and sequence. Bottom: Pol II and ATF4 were analyzed at sites 1, 3, and 4 by ChIP. The SKOV3 cells were treated with 2-DG for 24 hours before crosslinking for ChIP and analysis by qPCR (n ≥ 1). The primers used were as follows. Site 1: forward, 5′-GGG AAA GAG AGA GGA CAG GAG C 3′), and reverse, 5′-TCT GAC ACC CTC ATT AGA CCC TT-3′; site 3: forward, 5′-TCA TTC CCC GCT CTT CAT TC-3′, and reverse, 5′-CAG AGA CTA TGT GAG ACA ATG GAG G-3′; site 4: forward, 5′-AAC CCA TGA AAC TTG TAC CCA A-3′, and reverse, 5′-GCT TAT CTA ATT CAA TCA AGG ACA G-3′. Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Analysis of c-Myc mRNA in SKOV3 cells. Cells were treated with 2-DG for the indicated time periods, and c-Myc expression was analyzed by qRT-PCR (n = 3). *P≤.05. Fertility and Sterility 2014 101, 1450-1457.e4DOI: (10.1016/j.fertnstert.2014.01.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions