Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: Human U87 and murine PPQ glioma cells treated with IgG control, DX1, or 4H2 were immunostained to detect antibody penetration and counterstained with DAPI. No significant uptake of IgG control was observed in either cell line. DX1 and 4H2 penetrated and localized into the nuclei and cytoplasm of cells, respectively. Representative merged images are shown. Bar = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Control

Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: ( a-c ) The effect of supplemental nucleoside on cellular penetration by DX1 and 4H2 into PPQ cells was determined by ImageJ quantification of fluorescence signal. ADE supplementation increased the efficiency of cellular penetration by DX1 by a factor of 1.70±0.01 (****P<0.0001) ( a ) but did not enhance 4H2 penetration ( b ). In contrast, GUO supplementation increased 4H2 cellular penetration by factor of 3.20±0.30 (****P<0.0001) ( c ), correlating with the known affinity of 4H2 for GUO. ( d ) 4H2 penetrates lung adenocarcinoma cells and localizes into cytoplasm. Representative images of control and 4H2-treated Cal12T cells immunostained for antibody penetration and counterstained with DAPI are shown. ( e ) Lysates of Cal12T cells treated with 0-1 mg/mL 4H2 for 24 hours were analyzed by western blot probed with primary actin antibody for loading control and an anti-mouse secondary antibody to detect the actin primary and 4H2 (both murine). 4H2 HC and LC ran at their expected MWs, showing the antibody is not significantly degraded 24 hours after cellular penetration. ( f ) Visualization of live A549 lung adenocarcinoma cells treated with FITC-labeled 4H2 and MitoTracker Red FM and Hoechst counterstains show 4H2 penetration into cytoplasm. Images of Cal12T cells from this experiment are shown in Supp. Fig. 1b . ( g ) Representative images of Cal12T cells treated with 4H2 immunostained for 4H2 (green) and markers of specific organelles (red), including EEA1 for endosomes, LAMP1 for lysosomes, COX IV for mitochondria, and RCAS1 for Golgi apparatus), and DAPI nuclear counterstained (blue). 4H2 signal did not specifically co-localize with any of the organelles, including endosomes and lysosomes. Error bars = SEM. Scale bars = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Fluorescence, Control, Western Blot, Labeling

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