Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. Immunohsitochemical localization of Ki67 in the uterine sections of Msx 1 f/f Msx 2 f/f (left panel, a and c) and Msx 1 d/d Msx 2 d/d (right panel, b and d) mice on day 4 of pregnancy. Panels a and b indicate lower magnification (20×) and c and d indicate higher magnification (40×). L and G indicate luminal epithelium and glandular epithelium respectively. B. Real-time PCR was performed to analyze the expression of glandular factors, Lif , Foxa2 and Spink3 in uteri of Msx 1 f/f Msx 2 f/f and Msx 1 d/d Msx 2 d/d mice on day 4 of pregnancy. The level of Ck18 was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, ***p<0.0001. C. Transmission electron microscopy of uterine sections obtained from Msx1 f/f Msx2 f/f (left panel, a and b) and Msx1 d/d Msx2 d/d (right panel, c and d) mice on day 4 of pregnancy. Panels a and c indicate lower magnification (5Kx) and b and d indicate higher magnification (30Kx). D. Immunohistochemical analysis of Muc-1 expression in the uterine sections of Msx 1 f/f Msx 2 f/f (upper panel) and Msx 1 d/d Msx 2 d/d (lower panel) mice on day 1 (a and d), day 4 (b and e) and day 5 (c and f) of pregnancy. L indicates luminal epithelium.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transmission Assay, Electron Microscopy, Immunohistochemical staining

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. The level of p-FRS2 was examined in the uterine sections of Msx1 f/f Msx2 f/f (upper panel) and Msx1 d/d Msx2 d/d (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. B. The level of p-ERK was examined in the uterine sections of Msx1 f/f Msx2 f/f (upper panel) and Msx1 d/d Msx2 d/d (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. L, G and S indicate luminal epithelium, glandular epithelium, and stroma respectively. C. FGFR-specific inhibitor PD173074 was applied to one uterine horn of Msx1 d/d Msx2 d/d (n = 3) mice on day 3 of pregnancy. The other horn served as vehicle-treated control. Uterine horns were collected on day 4 morning and sections were subjected to immunohistochemistry to detect p-FRS2, Ki67, and Muc-1.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Immunohistochemistry

Journal: Reproductive Sciences

Article Title: Characterization of Molecular Changes in Endometrium Associated With Chronic Use of Progesterone Receptor Modulators: Ulipristal Acetate Versus Mifepristone

doi: 10.1177/1933719117746764

Figure Lengend Snippet: Downregulation of fibroblast growth factor (FGF) 18 expression in response to mifepristone in human endometrial stromal cells. Human endometrial stromal cells were subjected to differentiation in response to 0.5 mM 8-bromo-cAMP, 1 μM progesterone (P), 10 nM 17β-estradiol (E), and 10 μM ulipristal acetate (UPA) or mifepristone for 6 days. Upper: Total RNA was isolated and subjected to quantitative polymerase chain reaction (qPCR) using primer for FGF18. Y axis indicates fold induction. The level of Rplp0 was used as an internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM from 3 separate samples. Lower: Immunocytochemical analysis of FGF18 expression in endometrial stromal in the absence of UPA or mifepristone (left panel), in the presence of UPA (middle panel), and in the presence of mifepristone (right panel). Representative images are shown.

Article Snippet: Fluoromount-G with 4′, 6-diamidino-2-phenylindole (DAPI) was purchased from eBiosciences, Hatfield, PA. Endometrial sections or endometrial stromal cells were incubated with 1 or more of the following primary antibodies: HAND2 (1:250, Santa Cruz Biotechnology, Santa cruz, CA; SC-9409), FGF18 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA; SC-393471), PR (1:100, DAKO, Denmark; DAKO-A0098), and Progesterone receptor B isoform (PRB) 22 (1:300, Cell Signaling, Beverly, MA; CST-31575).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Control, Gene Expression

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: Real-time PCR was performed to monitor the expression of mRNAs corresponding to Msx1 and Msx2 in uterus on days 1 to 5 of gestation. The relative levels of gene expression on different days of pregnancy were determined by setting the expression level of Msx1 mRNA (A, Left panel) and Msx2 mRNA (B, Left panel) on day 1 of pregnancy at 1.0. Rplp0 , encoding a ribosomal protein, was used to normalize the level of RNA. Uterine sections from day 1 to day 5 (a–e) of pregnancy were subjected to immunohistochemical analysis using anti-MSX1 (A, Right panel) and anti-MSX2 (B, Right panel) antibodies. Panel f shows uterine sections from day 3 pregnant mice treated with non-immune IgG. L, G and S indicate luminal epithelium, glandular epithelium and stroma, respectively.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Immunohistochemical staining

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: Ablation of uterine Msx 1 and Msx 2 leads to female infertility.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques:

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. Uterine sections obtained from Msx 1 f/f Msx 2 f/f (left panel) and Msx 1 d/d Msx 2 d/d (right panel) mice on day 4 of pregnancy were subjected to IHC using antibodies against PGR (top panel, a and b), ESR1 (middle panel, c and d) and phospho-ESR1 (lower panel, e and f). B. Real-time PCR was performed to analyze the expression of E-regulated genes, lactotransferrin ( Ltf ), Clca3, lipocalin2 and Muc-1 in uteri of Msx 1 f/f Msx 2 f/f and Msx 1 d/d Msx 2 d/d mice on day 4 of pregnancy. The level of Ck18 was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, *p<0.05. C. Real-time PCR was performed to analyze the expression of P-regulated genes, Ihh, COUP-TF II, Hand2 and Hoxa10, in uteri of Msx 1 f/f Msx 2 f/f and Msx1 d/d Msx2 d/d mice on day 4 of pregnancy. The level of Rplp0 or Ck18 was used as internal control to normalize gene expression.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Gene Expression

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. Embryo implantation sites were examined in Msx1 f/f Msx2 f/f and Msx1 d/d Msx2 d/d mice by the vascular permeability assay, which can be scored as distinct blue bands (red arrows) following an injection of Chicago blue dye on day 5 of pregnancy (D5, n = 6) or direct eye-visualization of implanted embryo on day 6 (D6, n = 4) and on day 7 (D7, n = 4) of pregnancy. The graph represents the quantification of implantation sites in Msx1 f/f Msx2 f/f and Msx1 d/d Msx2 d/d mice on day 5 of pregnancy. B. Failure of embryo attachment in Msx1 d/d Msx2 d/d uteri. Histological analysis of uterine sections obtained from Msx1 f/f Msx2 f/f (a) and Msx1 d/d Msx2 d/d (b) mice on day 5 (n = 3) of pregnancy by Hematoxylin and Eosin staining. Note the intimate contact between embryo and luminal epithelium in Msx 1 f/f Msx 2 f/f mice and the free floating embryo in the uterine lumen of Msx 1 d/d Msx 2 d/d mice. L and E indicate luminal epithelium and embryo respectively.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Permeability, Injection, Staining

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. Immunohsitochemical localization of Ki67 in the uterine sections of Msx 1 f/f Msx 2 f/f (left panel, a and c) and Msx 1 d/d Msx 2 d/d (right panel, b and d) mice on day 4 of pregnancy. Panels a and b indicate lower magnification (20×) and c and d indicate higher magnification (40×). L and G indicate luminal epithelium and glandular epithelium respectively. B. Real-time PCR was performed to analyze the expression of glandular factors, Lif , Foxa2 and Spink3 in uteri of Msx 1 f/f Msx 2 f/f and Msx 1 d/d Msx 2 d/d mice on day 4 of pregnancy. The level of Ck18 was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, ***p<0.0001. C. Transmission electron microscopy of uterine sections obtained from Msx1 f/f Msx2 f/f (left panel, a and b) and Msx1 d/d Msx2 d/d (right panel, c and d) mice on day 4 of pregnancy. Panels a and c indicate lower magnification (5Kx) and b and d indicate higher magnification (30Kx). D. Immunohistochemical analysis of Muc-1 expression in the uterine sections of Msx 1 f/f Msx 2 f/f (upper panel) and Msx 1 d/d Msx 2 d/d (lower panel) mice on day 1 (a and d), day 4 (b and e) and day 5 (c and f) of pregnancy. L indicates luminal epithelium.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Gene Expression, Transmission Assay, Electron Microscopy, Immunohistochemical staining

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. Real-time PCR was performed to analyze the expression of Wnt ligands in uterine epithelial cells of Msx1 f/f Msx2 f/f and Msx1 d/d Msx2 d/d mice on day 4 of pregnancy. The level of Ck18 was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, *p<0.01, ***p<0.0001. B. Real-time PCR was performed to analyze the expression of Wnt ligands in uterine stromal cells of Msx1 f/f Msx2 f/f and Msx1 d/d Msx2 d/d mice on day 4 of pregnancy. C. Real-time PCR was performed to analyze the expression of Fgf family members in uterine stromal cells of Msx1 f/f Msx2 f/f and Msx1 d/d Msx2 d/d mice on day 4 of pregnancy. The level of Rplp0 was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, *p<0.01, **p<0.001, ***p<0.0001. D. The level of active β-catenin in uterine sections of Msx1 f/f Msx2 f/f (left panel) and Msx1 d/d Msx2 d/d (right panel) mice on day 4 of pregnancy was analyzed by IHC. (Magnification: a and c: 10×, b and d: 40×) E. Primary stromal cells were isolated from uteri of Msx1 d/d Msx2 d/d mice on day 3 of pregnancy and transfected with siRNA targeted to the β-catenin mRNA. Total RNA was isolated 24 h after transfection to analyze the expression of Fgf family members by Real-time PCR. The level of Rplp0 was used as an internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, *p<0.01, **p<0.001, ***p<0.0001.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Gene Expression, Isolation, Transfection

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: A. The level of p-FRS2 was examined in the uterine sections of Msx1 f/f Msx2 f/f (upper panel) and Msx1 d/d Msx2 d/d (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. B. The level of p-ERK was examined in the uterine sections of Msx1 f/f Msx2 f/f (upper panel) and Msx1 d/d Msx2 d/d (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. L, G and S indicate luminal epithelium, glandular epithelium, and stroma respectively. C. FGFR-specific inhibitor PD173074 was applied to one uterine horn of Msx1 d/d Msx2 d/d (n = 3) mice on day 3 of pregnancy. The other horn served as vehicle-treated control. Uterine horns were collected on day 4 morning and sections were subjected to immunohistochemistry to detect p-FRS2, Ki67, and Muc-1.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Immunohistochemistry, Control

Journal: PLoS Genetics

Article Title: Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

doi: 10.1371/journal.pgen.1002500

Figure Lengend Snippet: In normal pregnancy, MSX1 and MSX2 act to repress WNT and β-catenin signaling and inhibit FGF synthesis in the uterine stroma, thereby suppressing stromal-epithelial cross-talk. In the absence of MSX1 and MSX2, FGFs are induced, activating the epithelial FGFR-ERK1/2 pathway, and promoting epithelial proliferation. Activated ERK1/2 then phosphorylates epithelial ESR1. This triggers transcriptional activation of ESR1 and expression of its target genes, such as Muc-1 , which prevent the functional transformation of the luminal epithelium to receptive state, blocking embryo implantation.

Article Snippet: After washing with PBS for 15 min and the slides were incubated in a blocking solution for 1 h before incubating them in primary antibody overnight at 4°C with antibodies specific for MSX1 (Abcam, ab73883), MSX2 (Santa Cruz, sc-15396), MUC1 (Novus biological, NB120-15481), Ki67 (BD Pharmingen, 550609), ESR1 (Santa Cruz, sc-7207), p-ESR1 (Santa Cruz, sc-12915), PGR (Neomarkers MS-194-PO), HAND2 (Santa Cruz sc-9409), phospho-FRS2 (R&D systems AF5126) and active β-catenin (PY489, Developmental Studies Hybridoma Bank, Iowa City, IA 52242).

Techniques: Activation Assay, Expressing, Functional Assay, Transformation Assay, Blocking Assay

Journal: Molecular medicine reports

Article Title: Optimization and enrichment of induced cardiomyocytes derived from mouse fibroblasts by reprogramming with cardiac transcription factors.

doi: 10.3892/mmr.2017.8285

Figure Lengend Snippet: Figure 1. Candidate factors for cardiomyocyte induction. TTFs were transduced with individual lentiviruses expressing (A) GATA4 (B) MEF2C (C) TBX5 and (D) HAND2, and were then compared with untransduced TTFs, which were used as the control. Cells were immunostained with corresponding antibodies and DAPI. Scale bars, 100 µm. TTFs, tail‑tip fibroblasts; GATA4, GATA binding protein 4; MEF2C, myocyte‑specific enhancer factor 2C; TBX5, T‑box transcription factor 5; HAND2, heart‑ and neural crest derivatives‑expressed protein 2.

Article Snippet: The primary antibodies against GATA4 (1:100, cat. no. sc-25310), MEF2C (1:100, cat. no. sc-13268), TBX5 (1:100, cat. no. sc-376952), HAND2 (1:100, cat. no. sc-9409), α-myosin heavy chain (MHC, 1:100, cat. no. sc-20641) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), α-sarcomeric actinin (1:100, cat. no. BM0003; Boster Biological Technology, Wuhan, China) and cardiac troponin T (cTnT, 1:200, cat. no. MAB-0374; Fuzhou Maixin Biotechnology, Fuzhou, China) were applied without washing and incubated at 4 ̊C overnight.

Techniques: Transduction, Expressing, Control, Binding Assay