atcc 35303  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway

doi: 10.3390/ijms22168837

Figure Lengend Snippet: Endoglin silencing affects vessel sprout diameter and permeability. ( a ) Three-dimensional (3D) reconstruction of ECFC ctr-siRNA and ECFC Eng-siRNA to evaluate sprout diameter. In green, Actin F (Alexa 488), and in red, nuclei (TOPRO-3) are stained (scale bar, 150 µm). ( b ) When Eng is suppressed, sprouts are wider than in controls (*** p < 0.001). ( c ) Using a real-time impedance-based cell analyzer (iCELLigence system, ACEA Biosciences, San Diego, USA), ECFC ctr-siRNA and ECFC Eng-siRNA are analyzed in basal condition and under 20 ng/mL TNFα stimulation (gray and black lines, respectively). As shown by the quantification in ( d ), a significant difference between ECFC ctr-siRNA (white column) and ECFC Eng-siRNA (black column) is found 12 h after TNFα stimulation (* p < 0.05). ( e ) Immunofluorescence staining for VE-CAD in ECFC-ctr and ECFC Eng-siRNA with the absence of or in the presence of 20 ng/mL TNFα for 1 h (scale bar, 22 µm). ( f ) Quantification of the pictures in ( c ) does not show differences between control and Eng-siRNA in terms of VE-CAD membrane staining nor of fluorescence intensity in the presence of TNFα (* p < 0.05).

Article Snippet: Endoglin-specific siRNA (Eng-siRNA; sc-35302, Santa Cruz Biotechnology, CA, USA) was used to silence human Eng.

Techniques: Permeability, Staining, Immunofluorescence, Control, Membrane, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway

doi: 10.3390/ijms22168837

Figure Lengend Snippet: Endoglin silencing affects F-actin polymerization. ( a ) Immunofluorescence of F-actin (green, Phalloidin-Alexa 488) in ECFC ctr-siRNA and Eng-siRNA (scale bar, 22 µm). ( b ) Quantification of ( a ) (*** p < 0.001). ( c ) Co-staining of F-actin (red, Phalloidin-Alexa 546) and tubulin (green) (scale bar, 33 µm). ( d ) Evaluation of F-actin vs. tubulin distribution, using Pearson’s coefficient (* p < 0.05).

Article Snippet: Endoglin-specific siRNA (Eng-siRNA; sc-35302, Santa Cruz Biotechnology, CA, USA) was used to silence human Eng.

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway

doi: 10.3390/ijms22168837

Figure Lengend Snippet: Endoglin silencing affects cofilin dynamics. ( a , b ) Western blot performed on n = 5 different clones of ECFC at passage 3–4, <30 days. A representative experiment is shown. Eng silencing in ECFC reduces P-Cofilin, suggesting an increased activity of cofilin when Eng is downregulated. Actin and tubulin were used as loading controls. The last row displays the reduced expression of Eng after siRNA silencing. ( b ) Quantification of five different ECFC clones shows a significant difference (*** p < 0.001) between ctr-siRNA and Eng-siRNA in the P-Cofilin/Total-Cofilin ratio. ( c ). Using a real-time impedance-based cell analyzer (iCELLigence system, ACEA Biosciences), ECFC controls (DMSO) and ECFC treated by LIMKi were analyzed in basal condition and under 20 ng/mL TNFα stimulation (gray and black lines, respectively). As shown by the quantification in ( d ), a significant difference between control ECFC (gray bar) and ECFC treated with LIMKi (black bar) 12 h after TNFα stimulation (** p < 0.01). ( e , f ) Immunofluorescence for F-actin in ECFC controls vs. ECFC Eng-siRNA +/−TNFα ( e ) and ECFC controls vs. ECFC LIMKi +/−TNFα ( f ). (scale bar, 22 µm). ( g ) The quantification of ( e ) and ( f ) confirms that ECFC Eng-siRNA and ECFC LIMKi display the same modifications in terms of F-actin (* p < 0.05).

Article Snippet: Endoglin-specific siRNA (Eng-siRNA; sc-35302, Santa Cruz Biotechnology, CA, USA) was used to silence human Eng.

Techniques: Western Blot, Clone Assay, Activity Assay, Expressing, Control, Immunofluorescence

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: BMP7 regulates endoglin in endometrial epithelial cells during their receptivity. (A) BMP7 expression was analyzed in endometrial epithelial cells by western blotting. (B) Through immunofluorescence, the localization of BMP7 was determined with a confocal laser-scanning microscope in endometrial epithelial cells of mouse origin. (C) Silencing of BMP7 from endometrial epithelial cells was confirmed by western blotting. The expression level of endoglin was examined in response to BMP7 silencing in endometrial epithelial cells by western blotting. (D) Endoglin was detected by western blotting in endometrial tissue protein extract from the day 5, 1000 h stage post-BMP7 silencing at day 4, 1000 h. (E) The transcript level of BMP7 was examined by reverse transcription-quantitative polymerase chain reaction in endometrial tissue at day 5 (1000 h) following BMP7 silencing. Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05. BMP, bone morphogenic protein; si, small interfering RNA.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Expressing, Western Blot, Immunofluorescence, Laser-Scanning Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: Endoglin expression was upregulated in the endometrium during the phases of receptivity for embryo implantation. (A) Endoglin was analyzed by western blotting in endometrial epithelial cells. (B) Endoglin expression was evaluated by western blotting in the endometrium during the endometrial (uterine) receptivity phases. (C) The transcript level of endoglin was also examined in the endometrium by reverse transcription-quantitative polymerase chain reaction during endometrial receptivity phases. Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: Endoglin inhibition causes poor implantation. (A) Endoglin inhibition during the pre-receptive stage of the endometrial receptivity period exhibited poor implantation and blastocyst numbers. (B) The expression level of endoglin was evaluated post-endoglin activity inhibition at day 5 (1000 h) in the endometrial tissue lysate. Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05. KD, knockdown.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Inhibition, Expressing, Activity Assay, Knockdown

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: BMP7 formed a complex with endoglin and Rac1 in the endometrium and Rac1 is regulated by endoglin. (A) Endoglin was immunopositive on the immunoblot of BMP7 IP samples from endometrial tissue. Western blotting revealed the presence of Rac1 on IP samples of BMP7 from endometrial tissue proteins. (B) Rac1 activity was evaluated in endometrial epithelial cells from the day 5, 0500 h stage. (C) Expression level of Rac1 was determined in endometrial epithelial cells from the day 5 (0500 h) stage in response to endoglin inhibition. (D) In the endometrium, following endoglin inhibition, Rac1 expression and activity were analyzed at implantation and non-implantation sites in the post-receptive stage (day 5, 1000 h). Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05; **P<0.01. BMP, bone morphogenic protein; IP, immunoprecipitation.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Western Blot, Activity Assay, Expressing, Inhibition, Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: GTPRac1 is influenced by BMP7 in endometrial epithelial cells during endometrial receptivity. (A) Rac1 activity in the GTP-bound state was analyzed in isolated and cultured endometrial epithelial cells during various stages of the endometrial receptivity period. (B) Expression of Rac1 was assayed in endometrial epithelial cells following BMP7 knockdown by siRNA. (C) Expression of Rac1 and activity were determined in the whole uterus/endometrium during the post-receptivity phase in response to BMP7 knockdown. Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05. BMP, bone morphogenic protein; siRNA, small interfering RNA; MO, Morpholino oligonucleotides.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Activity Assay, Isolation, Cell Culture, Expressing, Knockdown, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: BMP7 coordinates endometrial epithelial cell receptivity for blastocyst implantation through the endoglin pathway in cell lines and a mouse model

doi: 10.3892/etm.2019.7265

Figure Lengend Snippet: BMP7 and endoglin participate in blastocyst attachment reaction. (A) Attachment of human placenta origin JAr cell spheroids on endometrial epithelial cells (Ishikawa) was analyzed post-BMP7 silencing from endometrial cells and expressed as a percentage. The percentage spheroids adhered/attached was determined following 24-h incubation. (B) BMP7 knockdown efficiency was analyzed in Ishikawa cells by western blotting. (C) Following endoglin inhibition in Ishikawa cells, JAr cell spheroid attachment was analyzed and expressed as a percentage. (D) Endoglin levels were determined in Ishikawa cells post-endoglin inhibition by western blotting. Data represent the mean ± standard error of the mean of three independent experiments. *P<0.05. BMP, bone morphogenic protein; siRNA, small interfering RNA.

Article Snippet: A 10-min co-culture of spheroids was carried out in the presence of endoglin siRNA, BMP7 siRNA or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature prior to washing with DMEM.

Techniques: Incubation, Knockdown, Western Blot, Inhibition, Small Interfering RNA