Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: Neonatal CHDs are associated with elevated plasma leucine levels and dysregulation of the WDPCP/EMCN axis. (a) The analysis of leucine levels in plasma samples of healthy controls and neonatal CHD patients ( n = 10 in each category). (b) Western blot analysis of the protein levels of WDPCP, and (c) EMCN in plasma samples of healthy controls and neonatal CHD patients ( n = 3 in each category). (d) Detection of phospho‐p38 and (e) phospho‐ERK levels by ELISA in plasma samples of healthy controls and neonatal CHD patients ( n = 10 in each category) were quantified ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: High‐leucine diet in pregnant female mice triggers CHD‐like features in the neonatal mice. Pregnant female mice ( n = 5 in each group) were fed the standard diet or a high‐leucine diet. (a) The plasma leucine levels were determined in the neonatal mice (2 weeks old) from the normal diet (control) and the high‐leucine diet (model) groups. (b) 320‐slice spiral CT analysis of epicardial adipose tissue volume (EATV) in neonatal mice from the control and model groups. (c) Analysis of WDPCP and EMCN protein expression in neonatal plasma samples of the control and model groups. (d) Detection of phospho‐p38, and (e) phospho‐ERK levels in plasma samples of neonatal mice from the the control and model groups. (f) Plasma VEGF‐A and ANG2 levels were analyzed by ELISA in the control and model groups. (g) Hematoxylin & Eosin staining in the cardiac tissues of neonatal mice from the control and model groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: WDPCP overexpression and MAPK activation ameliorate high‐leucine‐induced CHDs. Pregnant female mice ( n = 5 in each group) were fed the standard diet or a high‐leucine diet. Neonatal mice from the high‐leucine diet group was administered an AAV‐carrying WDPCP expression construct or a MAPK signaling pathway activator (C16‐PAF) for 2 weeks. (a) Western blot analysis of WDPCP and EMCN protein levels in plasma samples of the neonatal mice in each group. (b) 320‐slice spiral CT analysis of epicardial adipose tissue volume (EATV) in the neonatal mice of each group. (c) Phospho‐p38 and (d). phospho‐ERK levels were examined in plasma samples of the neonatal mice by ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. AAV, adeno‐associated virus.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Over Expression, Activation Assay, Expressing, Construct, Western Blot, Enzyme-linked Immunosorbent Assay, Virus

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: High‐leucine levels regulate WDPCP/EMCN expression and the MAPK/ERK signaling, and impairs the EMT in HCMECs. HCMECs were cultured in standard medium (control, 0.8 mM) or high‐leucine medium (4 mM) for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in the control and high‐leucine conditions. (b) Phospho‐p38 and (c). phospho‐ERK levels were examined in HCMECs cultured under the control and high‐leucine conditions. (d) Wound healing assay and (e) transwell invasion assay in HCMECs cultured under the control and high‐leucine conditions. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and an epithelial marker (E‐cadherin) in HCMECs cultured under the control and high‐leucine conditions. (g) CCK‐8 cell proliferation assay of HCMECs under the control and high‐leucine culturing conditions. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Expressing, Cell Culture, Control, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker, CCK-8 Assay, Proliferation Assay

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: WDPCP and MAPK signaling mediates the suppressive effect of high‐leucine levels on the EMT and cell migration in HCMECs. HCMECs transfected with a WDPCP expression vector or treated with the MAPK activator were cultured in standard medium (control) or high‐leucine medium for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in each experimental group. (b) Phospho‐p38 and (c) phospho‐ERK levels were examined in HCMECs of indicated experimental groups. (d) Wound healing assay and (e) transwell invasion assay in HCMECs of indicated experimental groups. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and an epithelial marker (E‐cadherin) in HCMECs of indicated experimental groups. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Migration, Transfection, Expressing, Plasmid Preparation, Cell Culture, Control, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: EMCN is a leucine‐induced effector negativity regulating the EMT and migratory ability of HCMECs. HCMECs transfected with the control siRNA (si‐NC) or an siRNA targeting EMCN (si‐EMCN) were cultured in a standard medium (control) or high‐leucine medium for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in each experimental group. (b) Phospho‐p38 and (c) phospho‐ERK levels were examined in HCMECs of indicated experimental groups. (d) Wound healing assay and (e) transwell invasion assay in HCMECs of indicated experimental groups. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and epithelial marker (E‐cadherin) in HCMECs of indicated experimental groups. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOne™ ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Transfection, Control, Cell Culture, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker