Journal: Annals of Surgery

Article Title: Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-mediated ARDS

doi: 10.1097/sla.0000000000004644

Figure Lengend Snippet: FIGURE 5. PMN depletion and SUCNR1 inhibition eliminates succinate/LPS mediated lung injury and SUCNR1 inhibition abrogates PMN sequestration. Panel A: As compared to 50 µM succi- nate/NS, 50 µM succinate/LPS caused increased EBD leak into the BALF. In rats pre-treated with an anti-rat neutrophil antibody, the neutrophil depletion eliminates succinate [50 µM]/LPS induced ALI, as measured by levels of EBD extravasation into BALF fluid, and the EBD extravasation is not significant compared to NS/NS in the PMN depleted group (*P < 0.05 compared to 50 µM succinate/LPS). Panel B: As com- pared to the HSA/ NS/NS (Control, left panel), HSA/succi-nate [50 µM]/NS (middle panel) caused PMN infiltration/ sequestration and an increase in the thickness of the pulmonary alveolar membranes. Pretreatment with the GPR91-2c inhibitor, GPR91-2c/ succi- nate [50 µM]/NS decreased the num- bers of the PMNs and decreased the thickness of the pulmonary alveolar membranes (right panel). The panel is representative of experiments per- formed in duplicate. The lung sections were stained with H&E and visualized at 40X. The bar graph is the histology score, membrane thickness of the his- tology. This figure represents the quan- tification of the pulmonary alveolar thickness of 10 images from 2 separate experiments, *=P < 0.05 versus the lungs from NS/ NS controls and †=P < 0.05 versus the lungs from succinate/NS treated rats. Panel C: Pretreatment for 30 min with the GPR91-2c [30 nM] inhibitor/NS did not elicit cause ARDS, as measured by EBD leak, in rats. Rats treated with the 1.25% HSA vehicle fol- lowed by 50 µM succinate/LPS or 500 µM succinate/LPS manifested ARDS. Pretreatment with the GPR91-2c abro- gated ALI caused by 50 µM succinate/ LPS or 500 µM succi-nate/LPS (*=P < 0.05 vs GPR91-2c/NS, GPR91-2c/LPS, HSA/50 µM succinate/ NS and HSA/500 µM succinate/NS; †=P < 0.05 vs HSA/50 µM succinate/LPS and HSA/500 µM succinate/LPS, n = 5 for each bar). ARDS indicates acute respiratory distress syn- drome; LPS indicates lipopolysaccharide; HSA, human serum albumin; PMN, neutrophils; SUCNR1, succinate receptor.

Article Snippet: PMNs were incubated with NS or succinate (500–1000 μM), fixed, smeared onto slides, incubated with a GPR91/ SUCNR1 antibody (Novus Biological, Littleton, CO), and then with a species-specific fluorescent antibody followed by microscopic imaging at 100X magnification.20,34 SUCNR1 Inhibitor Synthesis and Inhibition of

Techniques: Inhibition, Control, Staining, Membrane

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Hepatic succinate levels and SUCNR1 expression increased after liver ischemia–reperfusion injury (IRI) in mice. C57BL/6 mice were subjected to 60 min of liver ischemia followed by 1, 6, or 24 h of reperfusion as indicated. Control mice were sham-operated. ( A ) Succinate levels in liver tissues increased after IRI. ( B ) Succinate levels in serum increased after IRI. ( C ) Sucnr1 mRNA expression in liver tissues was upregulated after IRI ( n = 6). ( D ) Representative western blots of liver tissues show increased SUCNR1 protein levels after IRI ( n = 3). ( E ) Representative immunofluorescence images show the co-localization of SUCNR1- and CLEC4F-positive cells in liver tissues. The double-positive cells are located within the hepatic sinusoids (indicated by the white triangle) surrounding the central venous (indicated by the white asterisk). Proportions of SUCNR + CLEC4F + cells increased after IRI ( n = 3). Bar = 50 µm. Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Expressing, Control, Western Blot, Immunofluorescence

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 alleviated liver ischemia–reperfusion injury (IRI) and hepatocyte apoptosis. Wild-type (WT) and Sucnr1 −/− mice underwent 60 min of ischemia followed by 6 h of reperfusion. ( A ) Representative images of hematoxylin–eosin (HE)-stained liver sections. Liver damage was quantified using Suzuki’s scores ( n = 6).Bar = 200 µm. ( B ) Serum ALT levels reflect liver function and hepatocellular injury ( n = 6). ( C ) Intrahepatic apoptosis was measured by TUNEL assay. Representative fluorescent sections are shown (red spots with DAPI-stained blue background represent TUNEL-positive cells) ( n = 3). Bar = 200 µm. (D) Representative western blots of Cleaved caspase 3 and Caspase 3 in liver tissues after IRI, and relative protein expression ratio of Cleaved caspase 3 to Caspase 3 was evaluated in each group ( n = 3). ( E–G ) In vivo, hepatocytes were cultured without Kupffer cells (KCs) (the Control) or co-cultured with KCs isolated from Sucnr1 −/− mice (the Sucnr1 −/− group) and WT mice (the WT group) after IRI. The percentage of apoptotic hepatocytes ( E ) was increased under co-culture with KCs from WT mice after liver IRI and alleviated under co-culture with KCs from Sucnr1 −/− mice after liver IRI ( n = 3). Relative cell viability ( F ) and relative lactate dehydrogenase (LDH) activity ( G ) of hepatocytes under co-culture with KCs from WT mice and Sucnr1 −/− mice after liver IRI ( n = 6). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, In Vivo, Cell Culture, Control, Isolation, Co-Culture Assay, Activity Assay

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: The critical role of SUCNR1 on Kupffer cells (KCs) in liver ischemia–reperfusion injury (IRI). Wild-type (WT) and Sucnr1 −/− mice were injected with clodronate or vehicle 72 h before ischemia, and samples were harvested after 6 h of reperfusion. Naïve WT mice served as control without any treatment. ( A ) Representative images of hematoxylin–eosin (HE)-stained liver sections. Immunohistochemical (IHC) staining of CLEC4F shows the efficiency of KC depletion in the liver. ( B ) Suzuki’s scores ( n = 6). Bar = 200 µm. ( C ) Ratios (%) of CLEC4F-positive cells ( n = 3). ( D ) Serum AST values from WT and Sucnr1 −/− mice were measured after 6 h of reperfusion ( n = 6). Results are presented as mean ± SEM. ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Injection, Control, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits the activation of Kupffer cells (KCs) following liver ischemia–reperfusion injury (IRI). ( A) Expressions of CD40, CD80, and MHC II in KCs from wild-type (WT) and Sucnr1 −/− mice after liver IRI were analyzed by flow cytometry ( n = 3). ( B ) Expressions of F4/80, CLEC4F, and VSIG4 in liver tissues from WT and Sucnr1 −/− mice after liver IRI was evaluated through immunohistochemical staining ( n = 3). Scale bar = 200 µm. ( C ) Primary KCs were incubated with various concentrations of succinate or stimulated with LPS (1 μg/mL) for 6 h. The mRNA expression of KC markers ( F4/80, Clec4f, and Vsig4 ) was analyzed ( n = 5). ( D ) Primary KCs from WT and Sucnr1 −/− mice stimulated with succinate or LPS (1 μg/mL) for 6 h. Relative mRNA expression of KC markers ( F4/80, Clec4f, and Vsig4 ) was analyzed ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Activation Assay, Flow Cytometry, Immunohistochemical staining, Staining, Incubation, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits M1 polarization of Kupffer cell (KC) following liver ischemia–reperfusion injury (IRI). ( A ) Polarization of KCs was assessed by immunohistochemical staining for iNOS and Arg1 in Wild-type (WT) and Sucnr1 −/− mice with or without liver IRI ( n = 3). Bar = 200 µm. ( B ) Sucnr1 deficiency decreased the ratio (%) of M1 KCs (CD11c + CD206 − ) and increased that of M2 KCs (CD11c − CD206 + ) ( n = 3). ( C, D ) Relative mRNA expression of M1 markers ( iNOS and Cox2 ) and M2 markers ( Arg1 and Ym1 ) in WT and Sucnr1 −/− mice with or without liver IRI ( C ) ( n = 6) and in WT and Sucnr1 −/− KCs stimulated with succinate or LPS (1 μg/mL) for 6 h ( D ) ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits proinflammatory cytokine release following liver ischemia–reperfusion injury (IRI). (A) Expressions of cytokines (TNF-α, IL-6, IL-1β and IL-10) in liver tissues from WT and Sucnr1 −/− mice mice after liver IRI was analyzed by immunohistochemical staining ( n = 3). Scale bar = 200 µm. (B) Concentrations of cytokines (TNF-α, IL-6, IL-1β and IL-10) in liver tissues from WT and Sucnr1 −/− mice with or without liver IRI ( n = 6). (C) Relative mRNA expression levels of cytokines ( Tnf , Il6 , Il1b and Il10 )) genes in WT and Sucnr1 −/− KCs stimulated with succinate or LPS for 6 h ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: SUCNR1 mediated Kupffer cell (KC) polarization following liver ischemia–reperfusion injury (IRI) via the ERK/NF-κB signaling pathway. ( A ) Protein expression was detected by Western Blot taken from KCs in Wild-type (WT) and Sucnr1 −/− mice with or without liver IRI ( n = 3). ( B-D ) KCs from WT mice were pretreated with Compound 4C (GPR91 antagonist, 5 μM) and SCH772984 (SCH; ERK inhibitor; 10 μM) 24 h prior to stimulation with succinate (1 mM), and the expression of KC protein levels was detected by Western Blot ( B ) ( n = 3), and the mRNA expression of M1-related genes ( C ) and pro-inflammatory genes ( D ) were measured by real-time polymeric chain reaction ( n = 5). Hepatocytes were co-cultured with WT mice-derived KCs that pretreated with Compound 4C and SCH772984 before stimulation with succinate. The percentage of apoptotic hepatocytes ( E ) ( n = 3), cell viability of hepatocytes ( F ) ( n = 6), and LDH activity were determined ( G ) ( n = 6). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Expressing, Western Blot, Cell Culture, Derivative Assay, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes

doi: 10.1172/jci173214

Figure Lengend Snippet: Figure 1. SUCNR1 is expressed in islets and β cells. (A) Sucnr1 mRNA levels analyzed in subcutaneous white adipose tissue (scWAT), visceral WAT tissue (vWAT), and liver, pancreas, and muscle tissue from male mice by quantitative PCR (n = 3–4). (B) Immunohistochemical (IHC) staining of SUCNR1 in male human and male mouse pancreas sections, and chromogranin A IHC staining or H&E staining. Scale bars: 50 μm. (C) Analysis of Sucnr1 mRNA expression in α and β cells isolated by FACS from male rat islets (n = 4). (D) In silico study of SUCNR1 gene expression regulation by genomic sequences and specific human adult islet transcriptional factors, and single-nucleotide polymorphisms (SNPs) associated with T2D localized within or surrounding the SUCNR1 locus. TFBS, transcription factor binding site. Data are presented as mean ± SEM.

Article Snippet: A polyclonal primary antibody against SUCNR1 (NBP1-0086) was obtained from Novus Biologicals.

Techniques: Real-time Polymerase Chain Reaction, Immunohistochemical staining, Immunohistochemistry, Staining, Expressing, Isolation, In Silico, Gene Expression, Genomic Sequencing, Binding Assay

Journal: Journal of Clinical Investigation

Article Title: SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes

doi: 10.1172/jci173214

Figure Lengend Snippet: Figure 2. SUCNR1 levels in islets are dysregulated in obesity and T2D. (A) Sucnr1 mRNA expression in the entire pancreas of wild-type mice fed normal chow diet (NCD) and high-fat diet (HFD) and db/db mice on NCD (n = 4–5). (B) SUCNR1 and GLP1R mRNA expression in islets from healthy donors (n = 7) and donors with obesity (n = 3). (C) SUCNR1 protein levels in human islet lysates from healthy donors (n = 6) and donors with obesity (n = 3) and T2D (n = 7). (D) Linear correlations between the BMI of donors and SUCNR1 mRNA (n = 10) and protein (n = 16) expression. Data are presented as mean ± SEM. *P < 0.05 vs. control (Student’s t test in A and B, Kruskal-Wallis test with Dunn’s test for multiple comparisons in C, or Pearson’s correlation coefficient in D).

Article Snippet: A polyclonal primary antibody against SUCNR1 (NBP1-0086) was obtained from Novus Biologicals.

Techniques: Expressing, Control

Journal: Journal of Clinical Investigation

Article Title: SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes

doi: 10.1172/jci173214

Figure Lengend Snippet: Figure 3. The succinate/SUCNR1 axis enhances glucose-stimulated insulin secretion in β cells. (A) Succinate quantification in the conditioned medium (CM) of MIN6 cells cultured in low- or high-glucose conditions (n = 6). (B) Sucnr1 mRNA expression in MIN6 cells stimulated with different concentrations of glucose for 3 or 24 hours (n = 4). (C) SUCNR1 protein levels in EndoC-βH1 cells stimulated with different con- centrations of glucose for 24 hours (n = 6). (D) Insulin quantification in the CM of MIN6 cells stimulated with succinate or cis-epoxysuccinic acid (cESA) at 2.8 mM or 16.7 mM glucose (n = 4). (E) Insulin secre- tion in glucose-stimulated insulin secretion assays in EndoC-βH5 cells stimulated with 500 μM succinate or 50 μM cESA at 0 mM or 20 mM glucose, determined in the CM by ELISA (n = 4). (F) Insulin secretion in EndoC-βH5 cells incubated with a human-specific SUCNR1 antagonist (1 μM NF-56-EJ40) and 500 μM succinate or 50 μM cESA (n = 4). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal conditions; ###P < 0.001 vs. succinate (Student’s t test in A, ANOVA with Dunnett’s test for mul- tiple comparisons in B–E, or ANOVA with Tukey’s test for multiple comparisons in F).

Article Snippet: A polyclonal primary antibody against SUCNR1 (NBP1-0086) was obtained from Novus Biologicals.

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Clinical Investigation

Article Title: SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes

doi: 10.1172/jci173214

Figure Lengend Snippet: Figure 4. SUCNR1 activation in β cells induces proximal signaling and Ca2+ mobilization, and is dependent on Gq and PKC pathways in β cells. (A) Western blot analysis of several phosphoproteins in MIN6 cells stimulated with 500 μM succinate or 50 μM cESA at different time points (n = 4–5). (B) Ca2+ mobilization in MIN6 cells stimulated with glucose and succinate assessed by Fura-2 AM fluorometric ratio. Left: Representative trace and quantification of the response to sequential increases in succinate concentration before and after high glucose exposure (n = 4). Right: Representative trace and quan- tification of the response to succinate concentrations elevated similarly after high glucose exposure (n = 6). (C) Intracellular Ca2+ mobilization in perifused islets from wild-type C57BL/6 male mice assessed by Fura-2 AM fluorometric ratio. Left: Representative trace of 1 mM succinate’s effect on intracellular Ca2+ mobilization at a basal glucose concentration (2.8 mM), followed by exposure to 16.7 mM glucose (n = 7 islets from 3 mice). Right: Representative trace and quantification of 1 mM succinate’s effect on intracellular Ca2+ mobilization at 8 mM glucose (n = 7 islets from 2 mice). (D) Insulin secretion in EndoC-βH5 cells incubated with 500 μM succinate and 1 μM of PKC inhibitor Gö 6983 (n = 4). (E) Insulin secretion in EndoC-βH5 cells incubated with 500 μM succinate and 1 μM of Gq inhibitor FR900359 (n = 4). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, succinate vs. basal con- dition; #P < 0.05, ##P < 0.01, cESA vs. basal condition or an inhibitor vs. succinate (ANOVA with Dunnett’s test for multiple comparisons in A and B, paired Student’s t test in C, or ANOVA with Tukey’s test for multiple comparisons in D and E).

Article Snippet: A polyclonal primary antibody against SUCNR1 (NBP1-0086) was obtained from Novus Biologicals.

Techniques: Activation Assay, Western Blot, Concentration Assay, Incubation

Journal: Journal of Clinical Investigation

Article Title: SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes

doi: 10.1172/jci173214

Figure Lengend Snippet: Figure 5. SUCNR1 in β cells is required for preserving insulin secretion and glucose homeostasis in HFD-fed male mice. (A) IHC staining of SUCNR1 in pancreas sections of control and Sucnr1-βKO mice accompanied by serial H&E staining. Scale bars: 50 μm. (B) Body mass of control and Sucnr1-βKO mice under HFD for 8 weeks (n = 8–9). (C) Blood glucose levels in control and Sucnr1-βKO mice in fasted or random-fed conditions (n = 8). (D) Plasma insulin levels in control and Sucnr1-βKO mice in fasted or random-fed conditions (n = 7–8). (E) Morphometric analysis of control and Sucnr1-βKO mice by H&E staining (n = 3). Scale bars: 50 μm. (F) Morphometric analysis of control and Sucnr1-βKO mice by immunofluorescence staining with insulin and glucagon and counterstaining with DAPI (n = 3–4). Scale bars: 50 μm. (G) Intraperitoneal (i.p.) and oral glucose tolerance tests in control and Sucnr1-βKO mice (n = 6–7). Displayed are the blood glucose levels, AUC, plasma insulin (n = 5–6), and GLP-1 levels (n = 5). (H) Insulin tolerance test in control and Sucnr1-βKO mice (n = 6–8). (I) HOMA-IR for control and Sucnr1-βKO mice (n = 5). (J) Insulin secretion in isolated islets from control and Sucnr1-βKO mice stimulated with or without 1 mM succinate or 100 μM cESA at 2.8 or 16.7 mM glucose (n = 5 islet pools from 5–6 mice). Data are presented as mean ± SEM or as box-and-whisker plots indicating median, first and third quartiles, and maximum and minimum values. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice, comparing experimen- tal groups in orally administered mice, or in indicated pairwise comparisons; ##P < 0.01, ###P < 0.001 comparing experimental groups in i.p.-administered mice (Student’s t test in B, E, F, and I comparing 2 groups, or 2-way ANOVA with Bonferroni’s test for multiple comparisons in C, D, G, H, and J).

Article Snippet: A polyclonal primary antibody against SUCNR1 (NBP1-0086) was obtained from Novus Biologicals.

Techniques: Preserving, Immunohistochemistry, Control, Staining, Clinical Proteomics, Immunofluorescence, Isolation, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

doi: 10.3390/ijms222112048

Figure Lengend Snippet: Succinate, SUCNR1 and VEGF upregulation in gestational diabetic placentas. ( A ) Succinate quantification in GDM and matched normal placental lysates using a spectrophotometric kit ( n = 3). ( B , C ) Western blot of SUCNR1 and VEGF in whole tissue lysates from GDM ( n = 7) and matched normal pregnancies ( n = 9). ( D ) Immunohistochemistry staining of VEGF in placental tissue sections from GDM and normal pregnancy. Image is representative for 4 different placentas per group. Quantification of the images was performed with Fiji and the ratio of positive cells was calculated ( n = 4). ( E ) Pearson correlation analysis of SUCNR1 and VEGF protein abundance in normal placental tissue ( n = 9), ** p < 0.01, r = 0.85. Data in ( A – C ) were analyzed by unpaired t -test, * p < 0.05, data are shown as mean ± SD. Representative images and densitometry analysis are shown for ( B , C ).

Article Snippet: The cells were blocked in 10% 2nd antibody host serum with 4% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with SUCNR1 antibody (Novus Biologicals, Littleton, CO, USA).

Techniques: Western Blot, Immunohistochemistry, Staining, Quantitative Proteomics

Journal: International Journal of Molecular Sciences

Article Title: SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

doi: 10.3390/ijms222112048

Figure Lengend Snippet: SUCNR1 expression in human full-term placental endothelial cells. ( A ) ISH combined with immunofluorescence staining of human full-term placenta. Tissue sections were stained with a probe against SUCNR1 and antibody against VWF as a marker for endothelial cells. Nuclei were counterstained with DAPI. Three sections from each placenta were examined and the shown image is representative for 5 normal placentas ( n = 5). White arrows show co-expression of SUCNR1 and VWF. ( B , C ) Immunofluorescence staining of FpECAs and FpECVs, respectively, using antibody against SUCNR1 and DAPI for counterstaining. Images are representative for 3 different isolations ( n = 3). ( D ) Western blot of SUCNR1 in isolated placental arterial and venous cells from GDM and normal pregnancies ( n = 3). ( E ) Western blot of SUCNR1 in primary HUVECs cultured in normal or high glucose for 48 h ( n = 3). For ( D ), unpaired t -test was used between groups, * p < 0.05, data are shown as mean ± SD ( n = 3). For ( E ), paired t -test was performed, * p < 0.05, data are shown mean ±SEM. Representative images and densitometry analysis are shown for ( D , E ).

Article Snippet: The cells were blocked in 10% 2nd antibody host serum with 4% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with SUCNR1 antibody (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Immunofluorescence, Staining, Marker, Western Blot, Isolation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

doi: 10.3390/ijms222112048

Figure Lengend Snippet: Surface and intracellular expression of SUCNR1 in HUVECs. ( A ) Flow cytometry staining of SUCNR1 in HUVECs with or without prior permeabilization steps ( n = 3). ( B ) Immunofluorescence staining of SUCNR1 in non-permeabilized HUVECs labeled with plasma membrane markers (WGA: apical, and VE-Cadherin: basolateral sides) and DAPI. ( C ) Immunofluorescence staining of permeabilized HUVECs, using mitochondrial RFP, antibody against SUCNR1 and DAPI. For ( B , C ), images are representative for 5 different experiments ( n = 5).

Article Snippet: The cells were blocked in 10% 2nd antibody host serum with 4% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with SUCNR1 antibody (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Flow Cytometry, Staining, Immunofluorescence, Labeling, Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

doi: 10.3390/ijms222112048

Figure Lengend Snippet: Knockdown of SUCNR1 suppresses the angiogenic phenotype of HUVECs. ( A ) Representative histogram of flow cytometric analysis of DY-547 labeled positive control SiRNA at 24, 48 and 72 h. ( B ) Representative Western blot of SUCNR1 in non-transfected, control or SUCNR1 SiRNA-transfected HUVECs at 72 h post transfection. ( C ) Representative image for immunofluorescence staining of SUCNR1 in HUVECs transfected with either control or SUCNR1 SiRNA for 72 h. DAPI was used for counterstaining (scale bar 10 µm, objective 20×). ( D ) RT-PCR of VEGF gene expression in control or SUCNR1 SiRNA-transfected HUVECs treated with either vehicle or succinate for 4 h. ( E ) Transwell migration assay of control or SUCNR1 SiRNA-transfected HUVECs in response to vehicle or succinate after 16 h; representative images are shown (scale bar 200 µm, objective 10×). ( F ) Scratch assay in control or SUCNR1 SiRNA-transfected HUVECs treated with vehicle or succinate for 12 h; representative images are shown (scale bar 500 µm, objective 10×). D, E and F were analyzed by two-way ANOVA followed by Tukey’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.0001, data are shown as mean ±SEM ( n = 3-5).

Article Snippet: The cells were blocked in 10% 2nd antibody host serum with 4% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with SUCNR1 antibody (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Labeling, Positive Control, Western Blot, Transfection, Control, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Transwell Migration Assay, Wound Healing Assay