Journal: Nature Communications

Article Title: Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

doi: 10.1038/s41467-024-51579-0

Figure Lengend Snippet: a – d Human white preadipocytes were pretreated with vehicle (Veh) or 100 nM EDN3 and with or without inhibitors for 3 days, followed by a 12-day adipogenesis. a Schematic of pretreatment experiments. UCP1 mRNA ( b ) and protein ( c ) levels in mature adipocytes (EV and EV and EDNRB OE). d UCP1 mRNA levels in mature adipocytes (EV and EDNRB KO). e Cytosolic calcium levels in human white preadipocytes after 100 nM EDN3 treatment. f , g Intracellular cAMP levels ( f ) and oxygen consumption rate (OCR; g ) in human white preadipocytes after 6 hours of Veh or EDN3 treatment. Maximal OCR was quantified in the right panel. h – j EV and EDNRB OE preadipocytes were pretreated with Veh or EDN3 combined with or without melittin (ME) or YM254890 (YM) for 3 days, followed by a 12-day adipogenesis. UCP1 mRNA ( h ) and protein ( i ) levels in mature adipocytes pretreated with ME. j UCP1 mRNA levels in mature adipocytes pretreated with YM. k The protein levels in preadipocytes after 6 hr of Veh or EDN3 treatment. The quantification of protein bands was in the right panels. l The EPAC1 protein in EDNRB OE preadipocytes after 48 hr of EPAC1 siRNA treatment. m EDNRB OE preadipocytes with or without EPAC1 knockdown were pretreated with Veh or EDN3 for 3 days, followed by a 12-day adipogenesis. Protein levels in mature adipocytes. n EV or EDNRB OE preadipocytes were pretreated with Veh, 1 μM or 10 μM of PD98059 (PD-1 or PD-10) for 3 days and were differentiated to mature adipocytes. UCP1 and DIO2 mRNA levels in human white adipocytes. n = 3 biological replicates/group in ( b – e ), ( i ), ( k – n ); n = 4 biological replicates/group in ( f – h ), ( j ). Data are presented as mean ± SEM. P values were determined using unpaired two-tailed t tests: ( l ); one-way ANOVA with Tukey’s multiple-comparison test: ( b – d ), ( f – k ), ( m ), ( n ); two-way ANOVA with Bonferroni’s multiple-comparison test: ( e ). Source data are provided as a Source Data file. Panel ( a ) was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Primary antibodies for EDNRB (NBP130599, Novus Biologicals), UCP1 (ab23841, abcam), phospho-ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling), total ERK1/2 (#4695, Cell Signaling), phospho-CREB (Ser133) (#9198, Cell Signaling), total CREB (#9197, Cell Signaling), Phospho-PKA Substrate (#9624, Cell Signaling), PPARγ (GTX32803, GeneTex), FABP4/AP2 (GTX636995, GeneTex), EPAC1 (#4155, Cell Signaling), TUBULIN (#2146, Cell Signaling), VINCULIN (sc-25336, Santa Cruz) or ACTIN (MAB1501, Millipore) were applied in blocking buffer over night at 4°C.

Techniques: Knockdown, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

doi: 10.1038/s41467-024-51579-0

Figure Lengend Snippet: a , b Male mice were intraperitoneally injected by tamoxifen 5 times within 7 days to induce EDNRB KO in PDGFRα + preadipocytes (iKO) and were then housed at 5 °C for 7 days. a The mRNA levels of thermogenic genes in scWAT. CTL: n = 5, iKO: n = 6. b The protein levels of UCP1 in scWAT. The quantification of protein bands in right panel. n = 4 mice/group. c , d Control and iKO mice were housed at thermoneutral temperature (30 °C, TN) or cold (5 °C) for 7 days. Images of immunohistochemical stain for UCP1 proteins (UCP1 IHC; c ) and HE stain ( d ) in scWAT. High magnification images were in the right panels. Scale bar=100 μm. Two independent experiments were repeated with similar results, as shown in Supplementary Fig. a and . e The phosphorylated ERK and total ERK protein in scWAT of mice after 7 days of cold exposure. The quantification of protein bands in right panel. n = 3 mice/group. f , g PDGFRα + cell lineage-tracing control and EDNRB iKO mice were housed at thermoneutral temperature (30 °C, TN) or cold (5°C) for 7 days. f The immunofluorescent images from the section of scWAT. Scale bar=100 μm. g Quantification of GFP signal in each section of scWAT. CTL at TN: n = 6, iKO at TN: n = 6, CTL at Cold: n = 6, iKO at Cold: n = 7, 4 slides/mouse. h The mRNA levels of Ednrb, Pdgfrα and Ebf2 in SVF of scWAT from mice housed at TN or cold for 7 days. CTL at TN: n = 3, iKO at TN: n = 3, CTL at Cold: n = 3, iKO at Cold: n = 4. Data are presented as mean ± SEM. P values were determined using unpaired two-tailed t tests: ( a) , ( b ), ( e ); one-way ANOVA with Tukey’s multiple-comparison test: ( g ), ( h ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for EDNRB (NBP130599, Novus Biologicals), UCP1 (ab23841, abcam), phospho-ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling), total ERK1/2 (#4695, Cell Signaling), phospho-CREB (Ser133) (#9198, Cell Signaling), total CREB (#9197, Cell Signaling), Phospho-PKA Substrate (#9624, Cell Signaling), PPARγ (GTX32803, GeneTex), FABP4/AP2 (GTX636995, GeneTex), EPAC1 (#4155, Cell Signaling), TUBULIN (#2146, Cell Signaling), VINCULIN (sc-25336, Santa Cruz) or ACTIN (MAB1501, Millipore) were applied in blocking buffer over night at 4°C.

Techniques: Injection, Control, Immunohistochemical staining, Staining, H&E Stain, Two Tailed Test, Comparison

Journal: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 2 – Expression of immunoreactive ETA and ETB receptors in fluorogold-labeled trigeminal ganglion neurons. Animals received fluorogold (FG, 4% solution) injected s.c. into the upper lip. After 5–7 days, FG-injected rats were sacrificed and both TG removed and processed for ETA and ETB receptor immunohistochemistry. In panel A, images on the right depict FG-labeled TG neurons; middle images show ETA and ETB receptor staining and images on the left show expression of ETA and ETB receptors on some FG positive neurons (arrows on merged images). The asterisk indicates ETB receptor expression on satellite glial cells. Tissue sections incubated in the absence of primary or secondary antibodies did not display any fluorescence (not shown). Images are representative of at least 3 sections from each ganglion, taken from 3 to 4 distinct animals. Panel B illustrates the size–frequency histogram of ETA and ETB positive neurons within the entire trigeminal ganglion (n=1460 neurons).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Labeling, Injection, Immunohistochemistry, Staining, Incubation, Fluorescence

Journal: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 3 – Co-localization of ETA and ETB receptors with TRPV1. Sections were labeled for TRPV1 receptors (in red, left images) and ETA or ETB receptors (ETAR and ETBR, in green). Images on the right depict co-localization of TRPV1 receptors with ETAR and ETBR (arrows on merged images). Tissue sections incubated in the absence of primary or secondary antibodies did not display any fluorescence (not shown). Images are representative of at least 3 sections from each ganglion taken from 3 to 4 distinct animals. Panel B illustrates the percentage of neurons immunoreactive for TRPV1, ETA (ETAR) and ETB receptors (ETBR), as well as the percentage of TRPV1 positive neurons that also express ETAR and ETBR (n=1102 neurons).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Labeling, Incubation, Fluorescence

Journal: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 7 – Influence of ETA and ETB receptor antagonists on ET-1-induced nociceptive responses. BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, each at 10 nmol/site) were administered 30 min before ipsilateral ET-1 (10 pmol/site) injection into the upper lip (A) or TMJ (B). Each value represents the mean±s.e.m. of the total facial grooming time (A) or the sum of nociceptive behaviors (B) recorded from 6 to 8 animals. Asterisks denote P≤0.05 relative to the PBS plus ET-1-injected group (ANOVA followed by Bonferroni's test).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Injection

Journal: PLoS ONE

Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ET B receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

doi: 10.1371/journal.pone.0174119

Figure Lengend Snippet: Concentration-response curves show the effect of increasing concentrations of Sarafotoxin 6c (S6c) in the left anterior descending artery (LAD) upstream of the ligature, LAD downstream of the ligature, and non-ischemic septal coronary artery (SCA) after treatment with (A) IR and vehicle or (B) U0126. The results are shown as mean values ± SEM (n = 6–7), *** P < 0.001; two-way ANOVA with repeated measurements; LAD downstream vs. upstream. Concentration-response curves show the effect of increasing concentrations of endothelin-1 (ET-1) after ET B receptor desensitization and BQ788 treatment in the LAD upstream of the ligature, LAD downstream of the ligature, and non-ischemic SCA after treatment with (C) the vehicle or (D) U0126. The results are shown as mean values ± SEM (n = 4–7), *** P < 0.001; two-way ANOVA with repeated measurements; LAD downstream vs. upstream.

Article Snippet: To prevent nonspecific staining, sections were blocked with 2% donkey serum and 1% bovine serum albumin (BSA) in T-PBS for 1 h. Samples were incubated at 4°C overnight with primary mouse anti-phosphorylated ERK1/2 antibody (1:200, ab50011, Abcam, Cambridge, UK), sheep anti-endothelin ET B receptor antibody (1:250, alx-210-506a, Alexis Biochemicals, Nottingham, UK) or mouse anti-ET-1 antibody (1:100, ab2786, abcam) diluted in T-PBS.

Techniques: Concentration Assay

Journal: Oncology letters

Article Title: Prognostic values of EDNRB in triple-negative breast cancer.

doi: 10.3892/ol.2020.12012

Figure Lengend Snippet: Figure 1. Expression of EDNRB in TNBC and normal breast tissue. (A) Analysis of differential EDNRB expression between TNBC and normal breast tissues in the TCGA cohort. (B) Analysis of differential EDNRB expression between TNBC and adjacent normal tissues in the TCGA cohort. (C) Immunohistochemical staining intensity distribution of 99 cases of TNBC. (D) Immunohistochemical results of normal breast tissues and TNBC tissues at 10x40 high magnification. *P<0.01. EDNRB, endothelin B receptor; TNBC, triple‑negative breast cancer.

Article Snippet: Rabbit anti‐human EDNRB monoclonal antibody (cat. no. 31191; Signalway Antibody LLC, College Park, MD, USA), secondary antibody (cat. no. sp‐9001; OriGene Technologies, Inc., Rockville, MD, USA) and diaminobenzidine (DAB) chromogenic solution (cat. no. sp‐9001; OriGene Technologies, Inc.) were purchased.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncology letters

Article Title: Prognostic values of EDNRB in triple-negative breast cancer.

doi: 10.3892/ol.2020.12012

Figure Lengend Snippet: Figure 2. Kaplan‑Meier survival curve of EDNRB expression of patients in HNCH cohort. EDNRB, endothelin B receptor; HNCH, Henan cancer hospital.

Article Snippet: Rabbit anti‐human EDNRB monoclonal antibody (cat. no. 31191; Signalway Antibody LLC, College Park, MD, USA), secondary antibody (cat. no. sp‐9001; OriGene Technologies, Inc., Rockville, MD, USA) and diaminobenzidine (DAB) chromogenic solution (cat. no. sp‐9001; OriGene Technologies, Inc.) were purchased.

Techniques: Expressing

Journal: Oncology letters

Article Title: Prognostic values of EDNRB in triple-negative breast cancer.

doi: 10.3892/ol.2020.12012

Figure Lengend Snippet: Figure 4. Clinical value analysis of Models 1 and 2. (A) Time‑dependent receiver operating characteristic curve analysis of prediction model. (B) Decision curve analysis for Model 1 and Model 2 in prediction of DFS of TNBC at the 3‑year point. Model 1 consisted of N stage and NAC. Model 2 consisted of N stage, NAC and EDNRB expression. DFS, disease‑free survival; TNBC, triple‑negative breast cancer; N, lymph node; NAC, neoadjuvant chemotherapy.

Article Snippet: Rabbit anti‐human EDNRB monoclonal antibody (cat. no. 31191; Signalway Antibody LLC, College Park, MD, USA), secondary antibody (cat. no. sp‐9001; OriGene Technologies, Inc., Rockville, MD, USA) and diaminobenzidine (DAB) chromogenic solution (cat. no. sp‐9001; OriGene Technologies, Inc.) were purchased.

Techniques: Expressing