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

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: PDI decreases SOD1 monomers as well as multimers. a Lysates from HEK293A cells transfected with SOD1 and PDI were examined by Western blotting under nonreducing conditions. b, c PDI decreased the relative level of mSOD1 (G93A) protein (shown on Y-axis), and the decrease was observed for both monomers (b) and multimers (c). However, the PDI-induced decrease in SOD1 levels was accentuated for SOD1 monomers of both the wild-type and mutant (SOD1: WT wild-type, G93A G93A mutant, Moc mock-transfected). Values are mean + SEM, n =4; *P <0.01, **P <0.001

Article Snippet: HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: Overexpression of PDI decreases SOD1 monomers as well as multimers. a, b HEK293A cells were transfected with SOD1 and wild-type (WT) PDI or mutant (MT) PDI, and the change in SOD1 protein was examined by immunoblotting under nonreducing (a) and reducing (b) conditions. In mutant PDI, the four cysteine residues were mutated to serine (C36S, C39S, C383S, and C386S). Expression of PDI decreased total SOD1 protein. c Media of NSC-34 cells expressing SOD1 and/or PDI was monitored for secreted SOD1. SOD1 in the media was seen in the multimer form and was not significantly different in regardless of the presence of PDI. Values are mean + SEM, n =4; *P <0.01, **P <0.001

Article Snippet: HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: Over Expression, Transfection, Mutagenesis, Western Blot, Expressing