1. Stomatin-like protein 2 is involved in endometrial stromal cell proliferation and differentiation during decidualization in mice and humans 
Qian Feng, Zhuo-Ying Hu, Xue-Qing Liu, Xue Zhang, Xi Lan, Yan-Qing Geng, Xue-Mei Chen, Jun-Lin He, Ying-Xiong Wang, Yu-Bin Ding 
Reproductive BioMedicine Online 
Volume 34, Issue 2, Pages (February 2017)
DOI: /j.rbmo
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2. Figure 1
slp-2/Slp-2 expression pattern during early pregnancy on pregnancy day 1, 4, 5, 6 and 8 in mice. (A) Real-time quantitative polymerase chain reaction examination of endometrial slp-2 mRNA expression. ACTB was used as an internal control; (B) and (C) Western blot analysis of Slp-2 protein expression. ACTB was used as an internal control; (D) Slp-2 expression and localization in a mouse uterus during early pregnancy detected by immunohistochemistry. Positive expression results in a brown colour. The inset on the bottom left of each panel is a magnified image of the smaller inset. One-way analysis of variance was used to calculate the differences in mRNA and protein levels. The results are represented as mean ± SE. Bars labelled with different lowercase letters indicate that the data are significantly different between groups (P < 0.05). Each experiment was repeated at least three times with three to four per experimental group. D, day of pregnancy; GE, glandular epithelium; IS, implantation sites; IIS, interimplantation sites; LE, luminal epithelial; NC, negative control; PDZ, primary decidual zone; S, stroma; SDZ, secondary decidual zone; em, embryo. Scale bar, 200 µm.
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3. Figure 2
Endometrial slp-2/Slp-2 expression in a pseudopregnant mouse. (A) The localization and expression level of Slp-2 in pseudopregnancy detected by immunohistochemistry. Positive expression results in a brown colour. LE, luminal epithelial; Scale bar, 200 µm; (B) and (C) Western blot analysis of Slp-2 expression in a pseudopregnant mouse day1 (PD1), PD4, PD5, PD6 and PD8. One-way analysis of variance was used. The results are shown as the mean ± SE. (D) Real-time quantitative polymerase chain reaction examination of endometrial slp-2 mRNA expression on PD1, PD4, PD5, PD6 and PD8. ACTB was used as an internal control. One-way analysis of variance was used. The results are shown as the mean ± SE. Bars labelled with different lowercase letters indicate that the data are significantly different between groups (P < 0.05). Each experiment was repeated at least three times with three to four per experimental group. GE, glandular epithelium; LE, luminal epithelial; NC, negative control; PD, day of pseudopregnancy.
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4. Figure 3
Slp-2 expression under artificially induced decidualization in mice. (A) Morphology and (B) weight changes under artificially induced decidualization on day 4 of pseudopregnancy, with one side of the uterine horn infused with 10 µl of corn oil (Oil). The other side of the uterine horn served as the control (CON); (C) Rreal-time quantitative polymerase chain reaction confirmation of decidual marker dtprp mRNA expression. Student's t-test analysis was used. The results are shown as the mean ± SE. *** P < was considered statistically significant. ACTB was used as an internal control; (D) immunostaining of slp-2 antibody in the artificially induced deciduoma (right, Oil) and untreated control (left, CON). Positive expression results in a brown colour. dc, decidual cells; LE, luminal epithelial. Scale bar, 200 µm; (E) and (F) Slp-2 expression changes detected by Western blot under artificial decidualization . ACTB was used as an internal control. The results are shown as the mean ± SE. Each experiment was repeated at least three times. *** P < was considered statistically significant. CON, control; LE, luminal epithelial; S, stroma.
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5. Figure 4
SLP-2/SLP-2 expression in the human endometrium. (A) SLP-2 expression in human endometrial tissues in the proliferative phase (pro) (n = 3), early-secretory phase (early-sec) (n = 3), mid-secretory phase (mid-sec) (n = 3) and late-secretory phase (late-sec) (n = 3) determined by immunohistochemistry. PC, positive control; NC negative control. For details regarding the tissue samples see Supplementary Table S1. Positive expression results in a brown colour. dc, decidual zone; GE, glandular epithelium; S, stroma. Scale bar: 50 µm; (B) histogram summarizing the results of immunostaining. Both the intensity of SLP-2 antibody and the ratio of SLP-2-positive stromal cell/total cells are calculated in five randomly selected areas in each slide. Immunostaining intensity was evaluated as negative (0), weak (1), intermediate (2) or strong (3) by four independent observers in a blinded fashion; (C) real-time quantitative polymerase chain reaction and (D and E) Western Blot detection of SLP-2 and SLP-2 expression, respectively, in human isolated endometrial stromal cells throughout the menstrual cycle. ACTB was used as an internal control. The results are shown as the mean ± SE. One-way analysis of variance was used in calculating differences between groups. Bars labelled with different lowercase letters indicate that the data are significantly different between groups (P < 0.05). Each experiment was repeated at least three times.
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6. Figure 5
Slp-2 expression was increased in mouse uterine endometrial stromal cells under in-vitro decidualization and knock down of slp-2 decreased decidualization marker dtprp mRNA expression. (A) Analysis of slp-2 expression on mRNA and (B, C) protein levels in the primary cultured mouse uterine endometrial stromal cells under in-vitro decidualization with 10 nM of oestradiol-17β (E2), 1 µM of progesterone (P4) and 0.5 mM 8-Br-cyclic adenosine monophosphate (cAMP) (Sigma) in Dulbecco's Modified Eagle's Medium F-12 with 10% charcoal-stripped fetal bovine serum for 72 h. ACTB was used as an internal control. Student's t-test was used. **P < 0.01 and ***P < were considered statistically significant; (D) real-time quantitative polymerase chain reaction detection of dtprp mRNA levels among mouse uterine endometrial stromal cells with or without E2, P4 and cAMP treatment and E2, P4 and cAMP treatment with slp-2-small interfering RNAs. One-way analysis of variance was used. The results are shown as the mean ± SE. *** P < was considered statistically significant. Primary mouse uterine endometrial stromal cells culture experiments were repeated three to four times and were done each time in duplicate. SiRNA, small interfering RNAs.
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7. Figure 6
SLP-2 was increased in human endometrial stromal cells under in-vitro decidualization and knockdown of SLP-2 decreased decidualization marker IGFPB1 and prolactin (PRL) mRNA expression. (A) Real-time quantitative polymerase chain reaction confirmation of SLP-2 mRNA expression under in vitro decidualization via oestradiol (E2), progesterone (P4) and cyclic adenosine monophosphate (cAMP) treatment for 13 days. Student's t-test analysis was used. The results are shown as the mean ± SE. *** P < was considered statistically significant. ACTB was used as an internal control; (B) the impact of human SLP-2 knockdown on PRL mRNA and (C) IGFBP1 mRNA expression under in-vitro decidualization via E2, P4 and cAMP treatment for 13 days. One-way analysis of variance was used. The results are shown as the mean ± SE. *** P < were considered statistically significant. Primary human endometrial stromal cells culture experiments were repeated three to four times and were done each time in duplicate. ACTB was used as an internal control. SiRNA, small interfering RNAs.
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8. Figure 7
The effect of SLP2 knockdown on mouse uterine endometrial stromal cell (mESC) and human endometrial stromal cell (hESC) proliferation under in-vitro decidualization. The percentage of proliferating cells under in-vitro decidualization with or without SLP-2-small interfering RNA (siRNA) in primary mESC (A, B), hESC (C, D). Positive cell number was calculated using a two-sided Fisher's exact test. Proliferating ESCs were labelled with (red). Cell nuclei were stained with Hoechst (blue). ** P < 0.01 was considered statistically significant. * P < 0.05 was considered statistically significant. E2, oestradiol; EDU, 5-ethynyl-20-deoxyuridine; P4, progesterone.
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9. Figure 8
Knockdown of SLP-2 inhibited cell proliferation marker Cyclin D3 expression under in-vitro decidualization in mouse uterine endometrial stromal cell (mESCs) (A and B) and human endometrial stromal cell (hESCs) (C and D). Student's t-test analysis was used. The results represent the mean ± SE. ** P < 0.01 and *** P < were considered statistically significant. ACTB was used as an internal control.
Reproductive BioMedicine Online  , DOI: ( /j.rbmo )
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