Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells  Hisayuu Cho, M.D., Hidetaka Okada, M.D., Tomoko Tsuzuki, M.D., Akemi Nishigaki, Ph.D., Katsuhiko Yasuda, M.D., Hideharu Kanzaki, M.D.  Fertility and Sterility  Volume 99, Issue 1, Pages 248-255.e2 (January 2013) DOI: 10.1016/j.fertnstert.2012.08.056 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Induction of (A) HAND2, (B) FBLN1, and (C) PRL mRNA levels during MPA-induced decidualization in ESCs. ESCs were cultured in the presence of MPA (10−7 mol/L), E2 (10−8 mol/L) + MPA (10−7 mol/L), or vehicle (Control) for the indicated number of hours (h) or days (d). The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Values are significantly different versus control (*P<.05; **P<.01). Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of steroid hormones on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, various doses of MPA (10−11, 10−9, and 10−7 mol/L), E2 (10−8 mol/L), and/or RU-486 (10−6 mol/L) for 12 days. The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of four separate experiments with different cell preparations, and each value represents mean ± SEM; n = 4. Values are significantly different versus control (*P<.05; **P<.01). Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Effects of 8-Br-cAMP on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, E2 (10−8 mol/L), MPA (10−7 mol/L), and/or 8-Br-cAMP (0.5 mmol/L) for 3 days. (B) Effects of various progestins on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, P (10−7 mol/L), MPA (10−7 mol/L), norethisterone (NET; 10−7 mol/L), levonorgestrel (LNG; 10−7 mol/L), dienogest (DNG; 10−7 mol/L), or dexamethasone (DEX; 10−7 mol/L) in the presence of E2 (10−8 mol/L) for 3 days. The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of (A) four and (B) seven separate experiments with different cell preparations, and each value represents mean ± SEM; (A) n = 4 and (B) n = 7. Values are significantly different versus control (*P<.05; **P<.01). Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of silencing with siRNA on MPA-induced HAND2 or FBLN1 levels. ESCs were pretreated with HAND2 siRNA (HAND2-A or HAND2-B), FBLN1 siRNA (FBLN1-A or FBLN1-B), or nonsilencing RNA (Control) for 2 days and then exposed to E2 (10−8 mol/L) + MPA (10−7 mol/L) for 3 days. The mRNA levels of (A) FBLN1, (B) HAND2, and (C) 18S were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Values are significantly different versus control (*P<.05; **P<.01). (D) The protein levels of FBLN1, HAND2, and β-actin were analyzed by Western blot analysis. Total cell lysates were obtained from ESCs cultured with the above-described treatment. The protein levels of FBLN1 (E) and HAND2 (F) were quantified using ImageJ. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Columns and vertical bars represent the mean ± SEM for combined data of three separate experiments with different cell preparations; n = 3. Values are significantly different versus control (**P<.01). Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Real-time PCR quantification of EF-1α gene expression in ESCs. (A) Validation of the efficiency of amplification of the internal control (EF-1α). Using reverse transcriptase, cDNA was synthesized from 1 μg total RNA isolated from human ESCs. Serial dilutions of cDNA over a 105-fold range were amplified by real-time PCR using gene-specific primers. The most concentrated sample contained cDNA derived from 20 ng total RNA. EF-1α average Ct values were calculated for each cDNA dilution and plotted against log cDNA dilution (n = 3). All data were fit using least squares linear regression analysis. Real-time PCR efficiency (E%) for amplification was calculated as described in the materials and methods. (B) Effects of steroid hormones on EF-1α mRNA levels in ESCs. ESCs were cultured with vehicle (Control), MPA (10−7 mol/L), and E2 (10−8 mol/L) + MPA (10−7 mol/L) for 12 days. The y-axis indicates real-time PCR cycle threshold numbers (Ct values). Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. (C) The EF-1α mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to 18S mRNA levels. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Effects of silencing HAND2 or FBLN1 on IGFBP-1 and DKK1 expression. ESCs were pretreated with HAND2 siRNA (HAND2-A or HAND2-B), FBLN1 siRNA (FBLN1-A or FBLN1-B), or nonsilencing RNA (Control) for 2 days and then exposed to E2 (10−8 mol/L) + MPA (10−7 mol/L) for 3 days. The mRNA levels of (A) IGFBP-1 and (B) DKK-1 were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM, n = 3. Fertility and Sterility 2013 99, 248-255.e2DOI: (10.1016/j.fertnstert.2012.08.056) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions