Journal: Obstetrics & Gynecology Science

Article Title: Correlation of VEGF, HIF-1α, and MMP2 expression in placental villi among patients with recurrent spontaneous abortion

doi: 10.5468/ogs.24176

Figure Lengend Snippet: Relative mRNA levels of VEGF, HIF-1α, and MMP2 were quantified in placental villous tissue from both RSA patients and the control group. (A) mRNA expressions of VEGF, HIF-1α, and MMP2 were determined in the placental villous tissue using RT qPCR. GAPDH was used as an internal control. (B) mRNA levels were assessed by RT-PCR. VEGF, vascular endothelial growth factor; RSA, recurrent spontaneous abortion; HIF-1α, hypoxia-inducible factor-1α; MMP2, matrix metalloproteinase-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; RT qPCR, real-time quantitative polymerase chain reaction; RT-PCR, real-time polymerase chain reaction. * P <0.05 vs. control group.

Article Snippet: The polymerase chain reaction (PCR) reverse transcription into cDNA (PrimeScript TM RT Reagent kit; Takara Bio Inc., Shiga, Japan) and PCR amplification kits (SYBR ® Premix Ex TaqTM II; Takara Bio Inc.) were purchased from TaKaRa (Takara Bio Inc.).

Techniques: Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endoplasmic Reticulum Stress‐Induced triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Downregulation Exacerbates Platelet Activation and Myocardial Infarction in Patients With Coronary Artery Disease

doi: 10.1161/JAHA.124.041220

Figure Lengend Snippet: A , RT‐PCR detection of TREM2 mRNA in platelets and PBMCs. Monocyte‐specific marker CD14 was used to rule out contamination of white blood cells. Human 227 bp of TREM2, 357 bp of CD14, and 90 bp of β‐actin are amplified; mouse 260 bp of TREM2, 247 bp of CD14, and 174 bp of β‐actin are amplified. B , Platelets express TREM2 protein. The 293T cell line that does not express TREM2 was used as a negative control. The clone's name of anti‐TREM2 antibody used for Western blot was TREM2 [ EPR20243 ]. C , Expression of TREM2 in human platelets as detected by confocal microscopy. Platelets were stained with antibodies against TREM2 (rabbit IgG as isotype control) and platelet marker CD41(mouse IgG as isotype control) and then detected using Alexa 594 and Alexa 488‐labeled secondary antibodies, respectively. The clone's name of anti‐TREM2 antibody used for immunofluorescence was TREM2 (D8I4C). D , Decreased TREM2 mRNA expression in platelets from patients with SAP (n=12) and ACS (n=16) compared with healthy donors (n=19), as evaluated by qPCR. mRNA levels were normalized to β‐actin. E , Decreased TREM2 protein levels in platelets from patients with CAD. The typical Western blot result and statistical analysis of the same 19 healthy donors, 12 patients with SAP, and 16 patients with ACS as shown in D are provided. Characteristics of the study population ( D ) and ( E ) are summarized in Table . F , Platelets from patients with ACS express lower TREM2, which is reversely correlated with CRP‐induced P‐selectin release analyzed by flow cytometry (n=25 in each group). G , CRP‐induced platelet P‐selectin release in patients with ACS is reversely correlated with platelet TREM2 expression level. Platelets from the same 25 patients with ACS in panel ( F ) were stimulated by 0.1 μg/mL CRP, P‐selectin release was analyzed by flow cytometry. Each spot represents a different individual (Pearson r=−0.4810, P =0.0014). Characteristics of the study population ( F ) and ( G ) are summarized in Table . One‐way ANOVA followed by Tukey's multiple comparison test was performed in ( D ). Kruskal–Wallis test followed by Dunn's multiple comparison test was performed in ( E ) and ( F ). Pearson's correlation analysis was used in ( G ). ACS indicates acute coronary syndrome; CAD, coronary artery disease; CD62P, P‐selectin; CRP, collagen‐related peptide; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; qPCR, quantitative polymerase chain reaction; RT‐PCR, reverse transcription polymerase chain reaction; SAP, stable angina pectoris; and TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: Total RNA was extracted using Trizol Reagent (Invitrogen, NY) and reverse transcribed to cDNA using a reverse transcription polymerase chain reaction (PCR) kit (TaKaRa, Dalian, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Negative Control, Western Blot, Expressing, Confocal Microscopy, Staining, Control, Labeling, Immunofluorescence, Flow Cytometry, Comparison, Fluorescence, Real-time Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endoplasmic Reticulum Stress‐Induced triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Downregulation Exacerbates Platelet Activation and Myocardial Infarction in Patients With Coronary Artery Disease

doi: 10.1161/JAHA.124.041220

Figure Lengend Snippet: A , Predicted C/EBPα binding site in human TREM2 promoter. B , ChIP analysis of C/EBPα binding to the TREM2 promoter in Meg‐01 cells. PCR amplification of TREM2 promoter region containing C/EBPα binding motif from Meg‐01 cells after ChIP. Nonspecific lgG was used as a control. C , qPCR analysis of C/EBPα‐bound TREM2 promoter from Meg‐01 cells after ChIP. Data are normalized to the preimmunoprecipitation input for each sample and expressed as the fold change (n=3). D , Luciferase reporter assay in 293T cells revealed that C/EBPα binds to the ‐298 TTGCA ‐294 region of the TREM2 promoter to promote transcription, which is inhibited by CHOP and deletion of the ‐298 TTGCA ‐294 region (△TTGCA) (n=4). E , Increased CHOP and GRP78 expression in platelets from patients with SAP and ACS. Representative Western blot result and a summary of the data from 19 healthy donors, 12 patients with SAP, and 16 patients with ACS are provided. F , Increased mRNA expression of ATF4/6 and HSP60/A4 in platelets from patients with ACS as detected by qPCR. The mRNA level was normalized to β‐actin. Data from 19 healthy donors and 16 ACS patients were presented. G , Decreased TREM2 was accompanied by increased CHOP in platelets from patients with ACS. Representative immunofluorescence images and the summary (n=4) are shown. H , CHOP expression is negatively correlated with TREM2 protein expression in platelets. Samples from Figures and including 19 healthy subjects, 12 patients with SAP, and 16 patients with ACS were analyzed (n=47). Characteristics of the study population are provided in Table . Unpaired t test was performed in ( C ) and ( G ). Statistical analyses were performed using 1‐way ANOVA followed by Tukey's multiple comparison test in ( D ) and ( E ). Mann–Whitney test was performed in ( F ). Pearson's correlation was used in ( H ). ACS indicates acute coronary syndrome; ATF4/6, activating transcription factor 4/6; CAD, coronary artery disease; C/EBPα, CCAAT enhancer‐binding protein α; ChIP, , chromatin immunoprecipitation; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose‐regulated protein; HSP 60/A4, heat shock protein 60/heat shock protein family A (Hsp70) member 4; IP, immunoprecipitation; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; SAP, stable angina pectoris; and TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: Total RNA was extracted using Trizol Reagent (Invitrogen, NY) and reverse transcribed to cDNA using a reverse transcription polymerase chain reaction (PCR) kit (TaKaRa, Dalian, China).

Techniques: Binding Assay, Amplification, Control, Luciferase, Reporter Assay, Expressing, Western Blot, Immunofluorescence, Comparison, MANN-WHITNEY, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction