Requirement of heart and neural crest derivatives–expressed transcript 2 during decidualization of human endometrial stromal cells in vitro  Hisayuu Shindoh, M.D., Hidetaka Okada, M.D., Tomoko Tsuzuki, M.D., Akemi Nishigaki, Ph.D., Hideharu Kanzaki, M.D.  Fertility and Sterility  Volume 101, Issue 6, Pages 1781-1790.e5 (June 2014) DOI: 10.1016/j.fertnstert.2014.03.013 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effects of siRNA-mediated HAND2 silencing on MPA-induced HAND2. (A) ESCs were treated with HAND2 siRNA (HAND2-A or HAND2-B) or nonsilencing RNA (Control) on day 0 and day 6 and were cultured with E2 (10−8 mol/L) + MPA (10−7 mol/L) for 12 days with medium changes every 3 days. (B) HAND2 mRNA levels were analyzed with the use of real-time PCR and calculated after normalization to EF1α mRNA levels. The effect of control was assigned a potency of 100%. Results are combined data of six separate experiments with different cell preparations, and each value represents mean ± SEM; n = 6. *Values significantly different versus control (P<.01). (C) The protein levels of HAND2 and β-actin were analyzed by Western blot analysis. Total cell lysates were obtained from ESCs cultured with above-described treatment. (D) These protein levels were quantified with the use of ImageJ. The effect of 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 significantly different versus control (P<.01). (E) Immunofluorescence staining of HAND2 protein in ESCs treated with HAND2-A, HAND2-B, or control siRNA. EF1α = elongation factor 1α; ESC = endometrial stromal cell; HAND2 = heart and neural crest derivatives–expressed transcript 2; MPA = medroxyprogesterone acetate; PCR = polymerase chain reaction; siRNA = small interfering RNA. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of HAND2 silencing on cell morphology during decidualization. ESCs were cultured with the following agents for 12 days as described in Figure 1: (A) vehicle; (B) E2 (10−8 mol/L) + MPA (10−7 mol/L); (C) E2 + MPA with nonsilencing RNA; (D) E2 + MPA with HAND2-A siRNA; and (E) E2 + MPA with HAND2-B siRNA. (F) The protein levels of connexin-43 (CX43) and β-actin were analyzed with the use of Western blot. The effect of 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 significantly different versus control (P<.01). Abbreviations as in Figure 1. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effects of HAND2 silencing on mRNA levels of decidualization genes. ESCs were treated with HAND2 siRNA (HAND2-A or HAND2-B) or nonsilencing RNA (Control) for 12 days as described in Figure 1. The mRNA levels of (A) FBLN1, (B) PRL, (C) TIMP3, (D) DKK1, (E) IL-15, (F) SGK1, (G) FOXO1A, and (H) IGFBP5 were analyzed with the use of real-time PCR and calculated after normalization to EF-1α mRNA expression. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of six separate experiments with different cell preparations, and each value represents mean ± SEM; n = 6. *Values significantly different versus control (P<.01). DKK1 = dickkopf-1; FBLN1 = fibulin-1; FOXO1A = forkhead box O1A; IGFBP5 = insulin-like growth factor–binding protein 5; IL-15 = interleukin-15; SGK1 = serum glucocorticoid kinase 1; TIMP3 = tissue inhibitor of metalloproteinase 3; other abbreviations as in Figure 1. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of HAND2 silencing on FOXO1A during decidualization. ESCs were treated with HAND2 or control siRNA for 12 days as described in Figure 1. (A) The protein levels of FOXO1A and β-actin were analyzed with the use of Western blot. (B) The effect of 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 significantly different versus control: *P<.05; **P<.01. (C) Immunofluorescence staining of FOXO1A protein in ESCs treated with HAND2-A, HAND2-B, or control siRNA. Abbreviations as in Figures 1 and 3. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 The mRNA levels of (A) FBLN1, (B) PRL, (C) TIMP3, (D) DKK1, (E) IL-15, (F) SGK1, (G) FOXO1A, (H) IGFBP5, and (I) HAND2 during MPA-induced decidualization. ESCs were cultured in the presence of E2 (10−8 mol/L) + MPA (10−7 mol/L) or vehicle (Control) 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 three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Values significantly different versus control: *P<.05; **P<.01. DKK1 = dickkopf-1; EF1α = elongation factor 1α; ESC = endometrial stromal cell; FBLN1 = fibulin-1; FOXO1A = forkhead box O1A; HAND2 = heart and neural crest derivatives–expressed transcript 2; IGFBP5 = insulin-like growth factor–binding protein 5; IL-15 = interleukin-15; MPA = medroxyprogesterone acetate; PCR = polymerase chain reaction; SGK1 = serum glucocorticoid kinase 1; siRNA = small interfering RNA; TIMP3 = tissue inhibitor of metalloproteinase 3. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Effects of HAND2 silencing on PRL production. ESCs were treated with HAND2 siRNA (HAND2-A or HAND2-B) or nonsilencing RNA (Control) on day 0 and day 6 and were cultured with E2 (10−8 mol/L) + MPA (10−7 mol/L) for 12 days with medium changes every 3 days. ELISA was used to measure PRL concentrations in the culture medium during the last 3 days of a 12-day culture period. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of four separate experiments with different cell preparations, and each value represents mean ± SEM; n = 4. *Values significantly different versus control (P<.01). Abbreviations as in Supplemental Figure 1. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Effects of HAND2 silencing on cell proliferation. ESCs were treated with HAND2 siRNA (HAND2-A or HAND2-B) or nonsilencing RNA (Control) for 2 days. ESCs were incubated with cell proliferation reagent WST-8 for 1 hour, and absorbance values at 450 nm were measured. Results are representative data of three experiments with different cell preparations. Columns and vertical bars represent the mean ± SEM in quadruplicate wells. Abbreviations as in Supplemental Figure 1. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Effects of 8-Br-cAMP on mRNA levels in ESCs. ESCs were cultured with vehicle, 8-Br-cAMP (0.5 mmol/L), and/or MPA (10−7 mol/L) for 3 days. The mRNA levels of (A) HAND2, (B) FOXO1A, and (C) PRL were analyzed with the use of real-time PCR and calculated after normalization to EF-1α mRNA expression. Results are combined data of eight separate experiments with different cell preparations, and each value represents mean ± SEM; n = 8. Values significantly different versus control: *P<.05; **P<.01. Abbreviations as in Supplemental Figure 1. Fertility and Sterility 2014 101, 1781-1790.e5DOI: (10.1016/j.fertnstert.2014.03.013) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions