Expression of KRAS in the endometrium of early pregnant mice and its effect during embryo implantation  Xia Long, Min Zhang, Xuemei Chen, Junlin He, Yubin Ding, Cuizhen Zhang, Xueqing Liu, Yingxiong Wang  Reproductive BioMedicine Online  Volume 31, Issue 1, Pages 51-61 (July 2015) DOI: 10.1016/j.rbmo.2015.04.005 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 mRNA expression of KRAS in mice endometria. Eight mice were used in each group and the embryos of day 5, 6 and 7 were separated from the endometria. D5IS and D5IIS mean implantation sites and inter-implantation sites on D5. Each sample was analysed in triplicate. The KRAS mRNA concentrations were normalized relative to β-actin. Values expressed as mean ± SEM. No statistically significant differences were found when the data were analysed by one-way ANOVA and least significant difference analysis. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Protein expression of KRAS in mice endometria. The level of KRAS protein expression was examined using western blotting. Total protein (30 µg) was extracted from the endometria of pregnant (A) and pseudopregnant (B) mice from different groups. (C) KRAS protein expression at implantation sites (IS) and inter-implantation sites (IIS) on D5. There were eight mice in each group, and the embryos were separated from the uteri of day 5, 6 and 7 pregnant mice. Each sample was analysed in triplicate. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Immunohistochemical analysis of mouse uteri by staining for KRAS. (A) The location of the KRAS protein was determined using immunohistochemistry (IHC), where a yellowish-brown stain corresponds to a positive signal (200×). Scale bar = 100 µm, the bar size of the insets is 200 µm. NC = non-pregnant control; IS = implantation sites; le = luminal epithelium; ge = glandular epithelium; s = stroma; PDZ = primary decidual zone; SDZ = secondary decidual zone. (B) Histogram summarizing the results of immunostaining as described in Materials and methods. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Mouse uteri decidualization was successfully induced after being injected with oil. Experimental decidualization was induced on day 4 of pesudopregnancy by intraluminal oil injection in one horn, whereas the other horn without any injection served as an internal control. On day 8, the mice were killed, and uterine weights were recorded. (A) Representative photograph of the deciduoma-induced uteri on day 8; (B) fold changes indicate a comparison of weights between the infused (oil) and noninfused (control) uterine horns. Error bars represent the SEM. ***, values are significantly different (P < 0.005) between the infused and noninfused uterine horns. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Expression of HOX10 and KRAS in the endometrium during artificial decidualization. (A) HOXA10, as a decidual marker, was detected by immunohistochemical analysis in the deciduoma-induced endometrium; (B) expression of KRAS in the deciduoma-induced endometrium. le = luminal epithelium; ge = glandular epithelium; s = stroma. Scale bar = 100 µm; (C) histogram summarizing the results of immunostaining as described in Materials and methods. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Prolactin (PRL) secretion levels in decidualized and control endometrial stromal cells. PRL secretion levels in decidualized and control endometrial stromal cells were measured by enzyme-linked immunosorbent assay (ELISA) at 72 h after decidual treatment. Error bars represent the SEM. Statistical analysis = ***P < 0.005. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 The expression levels of the KRAS protein in decidualized and control endometrial stromal cells measured by western blotting at 72 h after decidual treatment. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 8 Down-regulation of KRAS expression inhibits stromal cell proliferation. (A) After transfecting with siRNA for 48 h, KRAS protein expression in the stromal cell was reduced; (B) stromal cells were transfected with 50 nmol/l of KRAS siRNA, NC siRNA, or nothing (control) at 48 h, and flow cytometric analysis revealed that the ratio of S phase stromal cells was lower after transfecting with KRAS siRNA (*P < 0.05). Experiments were repeated three separate times. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 9 KRAS function during stromal cells decidualization. Prolactin (PRL) secretion level in the non decidualized control stromal cells (control), decidualized stromal cells and KRAS knock-down decidualized stromal cells (siRNA) measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after decidual treatment. Statistical analysis = ***P < 0.005. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 10 Knock-down KRAS expression in mouse uteri. (A) The mRNA levels of KRAS on day 6 (48 h after injection) were examined by qPCR. Error bars represent the SEM. Statistical analysis = **P < 0.01; (B) KRAS protein on day 6 (48 h after injection) was examined by western blotting. LIP = lipofectamine 2000; NC = negative siRNA. Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 11 Effect of KRAS expression on mouse litter size. Representative uteri of mice on D6 are shown in (A) and (B). The red arrows correspond to the implantation sites and the black arrows correspond to the ovaries. LIP = lipofectamine 2000; NC = negative siRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2015 31, 51-61DOI: (10.1016/j.rbmo.2015.04.005) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions