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Cell Signaling Technology Inc rabbit polyclonal antibody against p57

Cell cycle regulators are affected by the delivery mode, and expressed in trophoblast organoids and placental tissues. ( A ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C <t>(p57)</t> was analyzed in placental tissues from different delivery modes ( n = 5). The results are presented as relative quantification (RQ) with minimum and maximum range. The mean value of the expression levels of SDHA (succinate dehydrogenase complex, subunit A), TBP (TATA box–binding protein), and YWHAZ (tyrosine 3–monooxygenase/tryptophan 5–monooxygenase activation protein, zeta polypeptide) served as the endogenous control. CS, caesarean section; eCS, emergency caesarean section after the onset of labor; VD, vaginal delivery; opVD, operative vaginal delivery. Unpaired Student’s t -test was used for statistical analysis, ** p < 0.01. ( B ) Representative immunohistochemistry-immunofluorescence (IHC–IF) image of a trophoblast organoid (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 50 µm. Inset scale: 10 µm. Villous cytotrophoblasts (CTBs) and the syncytiotrophoblast (STB) are indicated. ( C ) Representative IHC-IF image of placental tissue (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is presented. Scale: 10 µm. ( D ) Representative IHC–IF image of placental tissue (7 weeks of gestation) stained for E-cadherin (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 10 µm. Inset scale: 5 µm. CTBs ongoing to fuse to the STB (fCTBs), CTBs and the STB are indicated. ( E ) Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with p21 (first row), p–p21 (second row), p27 (third row) or p57 (fourth row) antibody (brown), respectively, and counterstained with hematoxylin (blue). Scale: 50 µm. Inset scale: 20 µm. EVTs (extravillous cytotrophoblasts) are indicated.

Rabbit Polyclonal Antibody Against P57, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia"

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

Journal: Cells

doi: 10.3390/cells10092214

Figure Legend Snippet: Cell cycle regulators are affected by the delivery mode, and expressed in trophoblast organoids and placental tissues. ( A ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed in placental tissues from different delivery modes ( n = 5). The results are presented as relative quantification (RQ) with minimum and maximum range. The mean value of the expression levels of SDHA (succinate dehydrogenase complex, subunit A), TBP (TATA box–binding protein), and YWHAZ (tyrosine 3–monooxygenase/tryptophan 5–monooxygenase activation protein, zeta polypeptide) served as the endogenous control. CS, caesarean section; eCS, emergency caesarean section after the onset of labor; VD, vaginal delivery; opVD, operative vaginal delivery. Unpaired Student’s t -test was used for statistical analysis, ** p < 0.01. ( B ) Representative immunohistochemistry-immunofluorescence (IHC–IF) image of a trophoblast organoid (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 50 µm. Inset scale: 10 µm. Villous cytotrophoblasts (CTBs) and the syncytiotrophoblast (STB) are indicated. ( C ) Representative IHC-IF image of placental tissue (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is presented. Scale: 10 µm. ( D ) Representative IHC–IF image of placental tissue (7 weeks of gestation) stained for E-cadherin (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 10 µm. Inset scale: 5 µm. CTBs ongoing to fuse to the STB (fCTBs), CTBs and the STB are indicated. ( E ) Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with p21 (first row), p–p21 (second row), p27 (third row) or p57 (fourth row) antibody (brown), respectively, and counterstained with hematoxylin (blue). Scale: 50 µm. Inset scale: 20 µm. EVTs (extravillous cytotrophoblasts) are indicated.

Techniques Used: Quantitative Proteomics, Expressing, Binding Assay, Activation Assay, Control, Immunohistochemistry, Immunofluorescence, Immunohistochemistry-IF, Staining

Cell cycle regulators are highly expressed during the first trimester of gestation. ( A – C ) Evaluation of positive cells in first trimester placental sections ( n = 6; named 6–9 week), early-onset control ( n = 20; named 25–33 week), and late-onset control samples ( n = 10; named 34–40 week). The results are presented as bar and scatter plots showing the mean value ± SD. ( A ) Quantification of p21 (left panel), p–p21 (middle panel), and p27 (right panel) positive CTBs in %. ( B ) Quantification of p21 (left panel), p-p21 (middle panel), and p27 (right panel) positive stained STB area in %. ( C ) Quantification of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) positive fCTBs in %. ( D ) The relative amount of the gene levels was analyzed from placental tissues: left panel CDKN1A (p21), middle panel CDKN1B (p27), and right panel CDKN1C (p57). The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student’s t -test or Mann–Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.

Figure Legend Snippet: Cell cycle regulators are highly expressed during the first trimester of gestation. ( A – C ) Evaluation of positive cells in first trimester placental sections ( n = 6; named 6–9 week), early-onset control ( n = 20; named 25–33 week), and late-onset control samples ( n = 10; named 34–40 week). The results are presented as bar and scatter plots showing the mean value ± SD. ( A ) Quantification of p21 (left panel), p–p21 (middle panel), and p27 (right panel) positive CTBs in %. ( B ) Quantification of p21 (left panel), p-p21 (middle panel), and p27 (right panel) positive stained STB area in %. ( C ) Quantification of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) positive fCTBs in %. ( D ) The relative amount of the gene levels was analyzed from placental tissues: left panel CDKN1A (p21), middle panel CDKN1B (p27), and right panel CDKN1C (p57). The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student’s t -test or Mann–Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.

Techniques Used: Control, Staining, Quantitative Proteomics, MANN-WHITNEY

p21 expression is reduced in fCTBs of early-onset PE samples. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 20) and patients with early-onset PE (ePE, n = 20) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed from placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Figure Legend Snippet: p21 expression is reduced in fCTBs of early-onset PE samples. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 20) and patients with early-onset PE (ePE, n = 20) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed from placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques Used: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Figure Legend Snippet: p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques Used: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O 2 ) or hypoxia (hypox, 1% O 2 ) for 48 h prior to RNA extraction or western blot analysis. ( A ) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( B ) Western blot analysis with cellular lysates from SGHPL-4 cells ( n = 3). GAPDH served as the loading control. ( C ) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( D ) Western blot analysis with cellular lysates from HTR cells ( n = 3). GAPDH served as the loading control. ( E ) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. ( F ) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results ( n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( G ) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated ( n = 5). GAPDH was used as the housekeeping gene control. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Figure Legend Snippet: p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O 2 ) or hypoxia (hypox, 1% O 2 ) for 48 h prior to RNA extraction or western blot analysis. ( A ) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( B ) Western blot analysis with cellular lysates from SGHPL-4 cells ( n = 3). GAPDH served as the loading control. ( C ) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( D ) Western blot analysis with cellular lysates from HTR cells ( n = 3). GAPDH served as the loading control. ( E ) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. ( F ) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results ( n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( G ) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated ( n = 5). GAPDH was used as the housekeeping gene control. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Techniques Used: Expressing, Isolation, RNA Extraction, Western Blot, Quantitative Proteomics, Control, Cell Culture

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Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: Cell cycle regulators are affected by the delivery mode, and expressed in trophoblast organoids and placental tissues. ( A ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed in placental tissues from different delivery modes ( n = 5). The results are presented as relative quantification (RQ) with minimum and maximum range. The mean value of the expression levels of SDHA (succinate dehydrogenase complex, subunit A), TBP (TATA box–binding protein), and YWHAZ (tyrosine 3–monooxygenase/tryptophan 5–monooxygenase activation protein, zeta polypeptide) served as the endogenous control. CS, caesarean section; eCS, emergency caesarean section after the onset of labor; VD, vaginal delivery; opVD, operative vaginal delivery. Unpaired Student’s t -test was used for statistical analysis, ** p < 0.01. ( B ) Representative immunohistochemistry-immunofluorescence (IHC–IF) image of a trophoblast organoid (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 50 µm. Inset scale: 10 µm. Villous cytotrophoblasts (CTBs) and the syncytiotrophoblast (STB) are indicated. ( C ) Representative IHC-IF image of placental tissue (8 weeks of gestation) stained for cytokeratin 7 (green), p21 (red), and nuclei (DAPI, blue) is presented. Scale: 10 µm. ( D ) Representative IHC–IF image of placental tissue (7 weeks of gestation) stained for E-cadherin (green), p21 (red), and nuclei (DAPI, blue) is shown. Scale: 10 µm. Inset scale: 5 µm. CTBs ongoing to fuse to the STB (fCTBs), CTBs and the STB are indicated. ( E ) Formalin-fixed and paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with p21 (first row), p–p21 (second row), p27 (third row) or p57 (fourth row) antibody (brown), respectively, and counterstained with hematoxylin (blue). Scale: 50 µm. Inset scale: 20 µm. EVTs (extravillous cytotrophoblasts) are indicated.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against GAPDH (GTX627408; GeneTex, Eching, Germany) and p53 (DO-1, sc-126; Santa Cruz, Heidelberg, Germany); rabbit monoclonal antibody against p21 (#2947) and p27 (#3686); and rabbit polyclonal antibody against p57 (#2557; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Quantitative Proteomics, Expressing, Binding Assay, Activation Assay, Control, Immunohistochemistry, Immunofluorescence, Immunohistochemistry-IF, Staining

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: Cell cycle regulators are highly expressed during the first trimester of gestation. ( A – C ) Evaluation of positive cells in first trimester placental sections ( n = 6; named 6–9 week), early-onset control ( n = 20; named 25–33 week), and late-onset control samples ( n = 10; named 34–40 week). The results are presented as bar and scatter plots showing the mean value ± SD. ( A ) Quantification of p21 (left panel), p–p21 (middle panel), and p27 (right panel) positive CTBs in %. ( B ) Quantification of p21 (left panel), p-p21 (middle panel), and p27 (right panel) positive stained STB area in %. ( C ) Quantification of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) positive fCTBs in %. ( D ) The relative amount of the gene levels was analyzed from placental tissues: left panel CDKN1A (p21), middle panel CDKN1B (p27), and right panel CDKN1C (p57). The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Unpaired Student’s t -test or Mann–Whitney U test referring to first trimester samples was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast.

Techniques: Control, Staining, Quantitative Proteomics, MANN-WHITNEY

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is reduced in fCTBs of early-onset PE samples. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 20) and patients with early-onset PE (ePE, n = 20) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57) was analyzed from placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. ( A – C ) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. ( A ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. ( B ) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. ( C ) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. ( D ) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. ( E ) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. ( F ) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t -test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Techniques: Expressing, Control, Staining, Western Blot, Quantitative Proteomics

Journal: Cells

Article Title: Functional Analysis of p21 Cip1/ CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia

doi: 10.3390/cells10092214

Figure Lengend Snippet: p21 expression is decreased in trophoblastic cell lines and in isolated primary trophoblasts under hypoxic conditions. Cells were grown under normoxia (norm, 21.4% O 2 ) or hypoxia (hypox, 1% O 2 ) for 48 h prior to RNA extraction or western blot analysis. ( A ) SGHPL-4 cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57) and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( B ) Western blot analysis with cellular lysates from SGHPL-4 cells ( n = 3). GAPDH served as the loading control. ( C ) HTR cells. Gene analysis of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) is shown. The results ( n = 3) are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( D ) Western blot analysis with cellular lysates from HTR cells ( n = 3). GAPDH served as the loading control. ( E ) Organoids (ORGs) cultured under normoxia (norm; n = 5) or hypoxia (hypox; n = 4). Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results are presented as relative quantification (RQ) with minimum and maximum range. GAPDH was used as endogenous control. ( F ) BeWo cells. Relative gene levels of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) are shown. The results ( n = 3) are presented as the relative quantification (RQ) with minimum and maximum range. GAPDH was used as the endogenous control. ( G ) Primary trophoblasts (pCTB) were isolated from healthy and preeclamptic (PE) donors. pCTBs from healthy donors were cultured under normoxia (norm; dot) or hypoxia (hypox, square), PE (triangle) pCTBs were grown under normoxia. The gene level of CDKN1A (p21), CDKN1B (p27), CDKN1C (p57), and TP53 (p53) was evaluated ( n = 5). GAPDH was used as the housekeeping gene control. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant; MV, mean value.

Techniques: Expressing, Isolation, RNA Extraction, Western Blot, Quantitative Proteomics, Control, Cell Culture

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