fugw perceval  (Addgene inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: ATP/ADP biosensor organoids for drug nephrotoxicity assessment

doi: 10.3389/fcell.2023.1138504

Figure Lengend Snippet: Reporter organoids with real-time biosensor Perceval HR to detect ATP/ADP ratio. (A) Bright-field (B.F.) and ATP/ADP ratio (lower) in nephron-like structures of 2D reporter organoids. (B) The channels of ATP (ex495/em540) and ADP (ex436/em540) are used for ratio calculation, transformed in artificial colors, and merged with the bright field. (C) Signal intensities at varied excitation wavelengths from 400 to 500 nm in the parental H9 organoids and Perceval H9 organoids in the normal and hypoglucose culture. (D) The signal ratios of em540 excited by 490 and 430 in the control and hypoglucose organoids, evaluated by a plate reader. The parental H9 was used as a control. (E) The reactivity of ATP/ADP ratio to toxic substance using NaN3. NaN3 was added at 0 min, and the same media were kept in the wells until 45 min. (F) 2D Reporter organoids before freezing, just after thawing, and 1 day after thawing.

Article Snippet: Lentivirus carrying Perceval HR was produced in HEK293 cells by transfection of FUGW-Perceval HR (addgene) ( ) and was transduced to H9.

Techniques: Transformation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: ATP/ADP biosensor organoids for drug nephrotoxicity assessment

doi: 10.3389/fcell.2023.1138504

Figure Lengend Snippet: Reporter organoids with real-time biosensor Perceval HR to detect ATP/ADP ratio. (A) Detection of nephrotoxicity by live monitoring of ATP/ADP ratio in 2D reporter organoids treated with 2.5 μg/mL AA and 5 µM cisplatin. (B) Representative images of ATP/ADP ratio monitoring in minimized 3D reporter organoids cultured on 384-well plates. The organoids were exposed to various toxicants (2.5 μg/mL AA, 5 or 50 µM cisplatin).

Article Snippet: Lentivirus carrying Perceval HR was produced in HEK293 cells by transfection of FUGW-Perceval HR (addgene) ( ) and was transduced to H9.

Techniques: Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: ATP/ADP biosensor organoids for drug nephrotoxicity assessment

doi: 10.3389/fcell.2023.1138504

Figure Lengend Snippet: Evaluation of the segment-specific injury by Perceval HR organoids. (A) Representative BF image and IF image of PODXL (red), LTL (cyan), and CDH1 (yellow) in 2D organoids. Scale bars: 200 μm. (B) Representative images for quantification of ATP/ADP ratio matched in position with the BF and IF images for segment identification in 2D organoids exposed to 5 μM cisplatin. Scale bars: 200 μm. (C) Detection of ATP/ADP ratio of each segment in 2D reporter organoids treated with hypoglucose, 5 and 50 μM cisplatin for 24 h n = 4 to 15. * p < 0.05.

Article Snippet: Lentivirus carrying Perceval HR was produced in HEK293 cells by transfection of FUGW-Perceval HR (addgene) ( ) and was transduced to H9.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Ribosomal biogenesis regulator DIMT1 controls β-cell protein synthesis, mitochondrial function, and insulin secretion

doi: 10.1016/j.jbc.2022.101692

Figure Lengend Snippet: DIMT1 and mitochondrial dysfunction in EndoC-βH1 cells. Representative immunoblots ( A ) and densitometric analyses ( B ) of mitochondrial complex protein I–V. Data are mean ± SD (n = 3). Glucose-induced (1 and 20 mM glucose) hyperpolarization of the inner mitochondrial membrane as shown by TMRM ( C ); average traces ( thick lines ) in cells treated with scramble siRNA compared to the difference in maximal hyperpolarization with FCCP in DIMT1-silenced cells ( thin lines ) are shown (scale bars: 20 μm) ( D ; data are mean ± SD; n = 3). E , cytosolic ATP/ADP ratios determined by Perceval HR ( green fluorescence ); average traces and the difference in the maximal rise in ATP/ADP ratio with oligomycin, (scale bars: 20 μm). ( F ; data are mean ± SD; n = 3). G , mitochondrial OCR is shown upon stimulation with 10 mM pyruvate as scramble cells ( black lines ) and as DIMT1-silenced cells ( gray lines ). The pyruvate-stimulated respiratory response, proton leak, ATP production and maximal mitochondrial respiration with FCCP and nonmitochondrial respiration (antimycin+rotenone) are each expressed as fold relative to basal ( H – M ; data are mean ± SD; n = 4). Statistical differences were compared by Student's t test and extreme studentized deviate test was used to determine outliers. ∗ p < 0.03 or <0.05 (as indicated), ∗∗ p < 0.01 and ∗∗∗ p < 0.001. DIMT1, dimethyladenosine transferase 1; TMRM, tetramethyl-rhodamine methyl ester.

Article Snippet: After 24 h, cells were cotransfected with 1 μg of plasmid encoding Perceval HR (Addgene ID:49,083) and 100 nM of DIMT1 siRNA at 50% cell confluency.

Techniques: Western Blot, Membrane, Fluorescence