Journal: Cell Communication and Signaling : CCS

Article Title: Human and viral chemokines differentially modulate G protein signaling, β-arrestin recruitment and chemotaxis mediated by the viral G protein-coupled receptor ORF74

doi: 10.1186/s12964-025-02546-9

Figure Lengend Snippet: Agonist and inverse agonist activities on β-arrestin1/2 recruitment to ORF74. The β-arrestin recruitment assays were performed using HEK293A cells co-transfected with pcDNA3.1(+)-ORF74-LgBiT and pEF1α-IRES SmBiT-ARRB1 or pEF1α-IRES SmBiT-ARRB2. Cells were stimulated with increasing concentrations of chemokines, and luminescence signals were measured for ( A ) β-arrestin1 and ( B ) β-arrestin2 recruitment. Data are presented as % of the maximal CXCL1 response set at 100%, with mean ± SD of three independent experiments, each performed in duplicate. Curves were fitted to a four-parameter logistic model

Article Snippet: HEK293A.ORF74 and HEK293A cells were detached with 0.25% Trypsin-EDTA (Thermo Fisher Scientific, #25200056), resuspended in growth medium and incubated at room temperature (RT) for 2 h. Then, cells were washed twice and resuspended in cold assay buffer [Hank’s Balanced Salt Solution (HBSS; Thermo Fisher Scientific), 20 mM HEPES buffer (Thermo Fischer Scientific), 0.5% FBS, pH 7.4].

Techniques: Transfection

Journal: Nature Communications

Article Title: In vivo measurement of an Apelin gradient with a genetically encoded APLNR conformation biosensor

doi: 10.1038/s41467-025-61781-3

Figure Lengend Snippet: Schematic illustration of the APLNR conformational biosensors design based on FRET ( a ), BRET ( b ) and cpGFP ( c ). mCitrine (FRET and BRET) and cpGFP were integrated at four different positions in the intracellular loop domain 3 of the APLNR (F233, R234, K235 and E236). d – f Functionality validation of the different biosensor variants stimulated with 10 µM Apelin in HEK293 cells. All four FRET biosensor variants show a significant FRET ratio decrease of around 10% after Apelin stimulation ( d ). In contrast, none of the BRET biosensors show a change in the BRET ratio after Apelin stimulation ( e ). All four cpGFP biosensor variants show a significant increase in the biosensor fluorescence intensity after Apelin stimulation ( f ). Data in ( d ) presented as mean ± StD from three transiently transfected single HEK293T cells. Data in ( e , f ) are presented as mean values ± SEM from three independent experiments conducted in transiently transfected HEK293A cells. Statistical analysis was performed by using a 2-way ANOVA followed by Šidák’s multiple comparison correction (* p < 0.0332; **** p < 0.0001). mTq2 mTurquoise2, FRET Förster resonance energy transfer, BRET bioluminescence resonance energy transfer, cpGFP circularly permuted GFP, HEK293 human embryonic kidney 293, n.s. not significant. Source data are provided as a Source Data file. a – c Created in BioRender. Schihada (2025) https://BioRender.com/e05s6eo .

Article Snippet: HEK293A (Cytion, 305070) and HEK293T (kind gift from Martin Lohse (Würzburg)) cells were cultured in DMEM (Gibco, 31966047) containing 10% FBS and 100 μg/ml penicillin-streptomycin mix (Gibco, 15140-122) and incubated at 37 °C with 5% CO 2 .

Techniques: Biomarker Discovery, Fluorescence, Transfection, Comparison, Förster Resonance Energy Transfer, Bioluminescence Resonance Energy Transfer

Journal: Nature Communications

Article Title: In vivo measurement of an Apelin gradient with a genetically encoded APLNR conformation biosensor

doi: 10.1038/s41467-025-61781-3

Figure Lengend Snippet: G i1 protein dissociation ( a ) and Arrestin3 recruitment ( b ) BRET assay of APLNR wildtype (WT), APLNR(F233)-cpGFP, and APLNR(K235)-cpGFP after Apelin stimulation. ng/ml represents the amount of receptor plasmid DNA (ng) used to transfect one ml of cells. c HEK293 cells were transiently transfected with Arrestin3-mCherry together with either APLNR WT, APLNR(F233)-cpGFP or APLNR(K235)-cpGFP. Cytoplasmic Arrestin3-mCherry localization is observed in unstimulated conditions. Apelin stimulation leads to a substantial translocation of Arrestin3-mCherry in APLNR WT co-transfected HEK293 cells. In contrast, only a minor fraction of Arrestin3-mCherry is membrane-localized when co-transfected with either APLNR(F233)-cpGFP or APLNR(K235)-cpGFP after Apelin stimulation. Arrowheads indicate membrane-localized Arrestin3-mCherry. Data are presented as mean values ± SEM from three independent experiments ( a ) and ±StD from four independent experiments ( b ) conducted in transiently transfected HEK293A cells ( a ) or transiently transfected HEK293T cells ( b , c ). cpGFP circularly permuted GFP, HEK293 human embryonic kidney 293. Source data are provided as a Source Data file.

Article Snippet: HEK293A (Cytion, 305070) and HEK293T (kind gift from Martin Lohse (Würzburg)) cells were cultured in DMEM (Gibco, 31966047) containing 10% FBS and 100 μg/ml penicillin-streptomycin mix (Gibco, 15140-122) and incubated at 37 °C with 5% CO 2 .

Techniques: Bioluminescence Resonance Energy Transfer, Plasmid Preparation, Transfection, Translocation Assay, Membrane

Journal: Nature Communications

Article Title: In vivo measurement of an Apelin gradient with a genetically encoded APLNR conformation biosensor

doi: 10.1038/s41467-025-61781-3

Figure Lengend Snippet: a , b Schematic illustration of the ratiometric APLNR conformational biosensors design. mScarlet-I3 and p2A-mScarlet-I3 were integrated C-terminally into the APLNR-cpGFP biosensors, resulting in membrane-bound ( a ) and cytosolic ( b ) mScarlet-I3 localization. c HEK293A cells were transiently transfected with the respective ratiometric biosensor plasmid and stimulated with Apelin. Stimulation with Apelin led to a strong increase in cpGFP over mScarlet-I3 fluorescence intensity of each ratiometric biosensor variant. d Quantification of the FRET ratio of the ratiometric biosensor with and without a p2A self-cleaving peptide exhibited no significant differences, indicating that cpGFP and mScarlet-I3 are no FRET partner. e Schematic illustration of the procedure. Plasmids encoding either UAS:APLNR(K235)-cpGFP-mScarlet-I3 or UAS:APLNR(K235)-cpGFP-p2A-mScarlet-I3 were injected into 1-cell stage Tg(fli1a:GAL4FF) zebrafish embryos and imaged at 28 hours post fertilization (hpf). f , g Quantification of APLNR(K235)-cpGFP over mScarlet-I3 fluorescence intensities in ISVs compared to the DA and PCV in UAS:APLNR(K235)-cpGFP-mScarlet-I3 ( g ) or UAS:APLNR(K235)-cpGFP-p2A-mScarlet-I3 ( h ) injected embryos at 28 hpf. Data in are presented as mean values ± SEM from three independent experiments conducted in transiently transfected HEK293A cells ( c , d ) or zebrafish embryos ( N number of embryos, n either ISVs or DA/PCV: g DA/PCV N / n : 32/32, ISV N / n : 32/110; h DA/PCV N / n : 25/25, ISV N / n : 25/75). Statistical analysis was performed by using ordinary One-way ANOVA, followed Dunnett’s multiple comparison correction ( d ) and two-tailed unpaired Student’s t -test with Welch’s correction ( g , h ). Scale bars 50 µm. ISV intersegmental vessel, DA dorsal aorta, PCV posterior cardinal vein, hpf hours post-fertilization, cpGFP circularly permuted GFP, HEK293 human embryonic kidney 293. Source data are provided as a Source Data file. a , b Created in BioRender. Schihada (2025) https://BioRender.com/odhtcw2 . e Created in BioRender. Schihada (2025) https://BioRender.com/mz23zor .

Article Snippet: HEK293A (Cytion, 305070) and HEK293T (kind gift from Martin Lohse (Würzburg)) cells were cultured in DMEM (Gibco, 31966047) containing 10% FBS and 100 μg/ml penicillin-streptomycin mix (Gibco, 15140-122) and incubated at 37 °C with 5% CO 2 .

Techniques: Membrane, Transfection, Plasmid Preparation, Fluorescence, Variant Assay, Injection, Comparison, Two Tailed Test