Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A & B : HEK-293 cells were transiently transfected with D 1 R::RFP and D 5 R::GFP and then grown on a cover slip or in a polarized state in a Transwell ™ insert; the colocalization of these receptors was evaluated via laser scanning confocal microscopy. Wheat germ agglutinin (WGA) tagged with Alexa Fluor□ 647 was used to visualized the plasma membrane, while DAPI (#H-1500, Vector Laboratories, Burlingame, CA) was used to visualized the nuclei. Colocalization was observed as discrete areas of white (red+green+blue) or yellow (red+green). Serial images along the X, Y, and Z axes were obtained in cells grown in Transwell ® and stitched together to show 3D colocalization of the receptors. 630x magnification, scale bar = 10 μm, n=3 independent experiments. C : hRPTCs were double-transfected with D 1 R and D 5 R tagged with the C and N termini of the fluorescent protein EYFP, respectively. The cells were grown on cover slips for 48 hours post-transfection, serum-starved for 2 hrs, and then prepared for confocal microscopy. The interaction of the tagged receptors results in the reconstitution and fluorescence of EYFP (pseudocolored green). An overlay of the BiFC signal and the nucleus (pseudocolored blue) is shown to indicate the distribution of the D 1 R-D 5 R complexes, 630x magnification, scale bar =10 μm, n=3 independent experiments. Transfection of D 1 R-EYFP alone was used as negative control.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A section of the human kidney was double immunostained for endogenous D 1 R (red) and D 5 R (green) ( A ). WGA (magenta) was used to visualize the plasma membrane, e.g., apical brush border of proximal tubules, while DAPI was used to visualize the nuclei. Colocalization in yellow is indicated by the arrows. DIC = differential interference microscopy. Sections of a mouse kidney infused with either vehicle (Basal) or fenoldopam was double immunostained for endogenous D 1 R (green) and D 5 R (red) ( B ). The RPT marker CD15 and DAPI were used to visualize the brush border and nuclei, respectively. Colocalization is indicated by the yellow or white areas in merged images. 630x magnification, scale bar=10 μm, n=3 independent experiments.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Microscopy, Marker

Journal: BMC neuroscience

Article Title: NPT100-18A rescues mitochondrial oxidative stress and neuronal degeneration in human iPSC-based Parkinson's model.

doi: 10.1186/s12868-025-00926-y

Figure Lengend Snippet: Fig. 1 Increased oxidative stress and mitochondrial dysfunction in patient-derived mDANs. (A) Schematic of experimental design. Fibroblasts from PD patients carrying a monoallelic SNCA locus duplication and unaffected control individuals were obtained through punch biopsy of the skin. Fibroblasts were reprogrammed into human iPSCs. Human iPSCs were first differentiated into NPCs through dual SMAD inhibition and activation of canonical Wnt signaling and then into mDANs using a FGF8b-based protocol. mDANs were treated with NPT100-18A during the entire differentiation and maturation periods. (B-D) Relative levels depicted as fold changes from the mean of control cell line C1 ± SD of n wells from N independent differentiations. Repli cates belonging to the same differentiation are shown in the same color. (B) CellRox Green fluorescence intensities (FIs) were assessed in living mDANs. CellRox and DAPI FIs were measured at 80 sites per well in 96-well plates using a CLARIOstar plate reader. CellRox FIs were normalized to respective DAPI FIs. Dots representing single well means (n = 15, N = 4). Right panels show representative microscopy images of CellRox and DAPI fluorescence signals. (C) Significantly reduced relative ATP levels measured in patient-derived compared to control mDANs. Relative ATP levels were assessed in mDAN lysates using a luciferase-based assay and a LUMIstar Omega plate reader. Values for single wells were normalized to the frequency of viable neurons (determined with a viability staining in mDANs cultured in parallel under the same conditions). Dots represent single wells (n = 9, N = 3). (D) MitoSOX Red FIs were measured in living mDANs analogously to (B). Dots represent single well means (n = 18, N = 4). Right panels show representative microscopy images of MitoSOX and DAPI fluorescence signals; for better visualization of neurons, β3-tubulin (TUBB3) was additionally immunofluorescently labelled. (B-D) Student’s t-test, *P < 0.05, ** P < 0.01, ***P < 0.001. Scale bar 50 μm in (B) and 20 μm in (D)

Article Snippet: CellROX, MitoSOX, and DAPI fluorescence intensities (FIs) were measured on a CLARIOstar Plus microplate reader (BMG Labtech) using the following excitation/emission wavelengths: CellROX – 500 − 20/530 − 25 nm; MitoSOX – 510 − 15/580 − 20 nm; DAPI – 360 − 20/460 − 30 nm.

Techniques: Derivative Assay, Control, Inhibition, Activation Assay, Fluorescence, Microscopy, Luciferase, Staining, Cell Culture