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: bioRxiv

Article Title: AalpiRNA-18529 regulates vitellogenesis of Aedes albopictus via the Gadd45a -mediated JNK-dependent nurse cell apoptosis pathway

doi: 10.1101/2024.08.20.608798

Figure Lengend Snippet: (A) Schematic diagram showing the relative position of mature Aalpi00018529 in the Ae. albopictus genome. Aalpi18529 originates from piRNA Cluster 215 (NW_021837156.1, 62116929-62144956) in scaffold_11 (NW_021837156.1, length 62,838,808 bp) and is located in exon 15 of the antisense transcript of a lncRNA (TCONS_00030668). (B) Temporal profiles of Aalpi-18529 and TCONS_00030668 at different developmental stages of Aedes albopictus determined by qRT‒PCR. E = n hours postoviposition embryo; 1 st -2 nd L= 1 st -2 nd instar larvae; 3 rd -4 th L = 3 rd -4 th instar larvae; P = pupae; F = adult female; M = adult male. The relative expression levels of Aalpi-18529 and TCONS_00030668 in 0-2 h postoviposition embryos were set as 1. (C) Spatial expression patterns of Aalpi-18529 and TCONS_00030668 in different tissues of Ae. albopictus adult females determined by qRT‒PCR. The relative expression levels of Aalpi-18529 and TCONS_00030668 in the head were set as 1. (D) Dynamic expression levels of Aalpi-18529 and TCONS_00030668 in the ovaries of adult females at different time points after a blood meal determined by qRT‒PCR. PE = hours postemergence; PBM = hours post blood meal. The relative expression levels of Aalpi-18529 and TCONS_00030668 in the 72 h PE groups were set as 1. (E) The relative expression quantities of Aalpi-18529 and TCONS_00030668 in the cell nucleus and cytoplasm fractions of adult females were measured by qRT‒PCR. AalRps7 and β-actin mRNA served as markers of cytoplasmic fractions, whereas U6 served as a marker of nuclear fractions. The X-axis indicates the different sample groups, and the Y-axis shows the relative expression levels. All qRT-PCRs were performed in triplicate with three biological replicates, and the values are presented as the means ± SEMs. (F) Schematics of the previtellogenic stage ovary and its ovarian ovariole (48 PE), and the major cellular types of the primary egg chamber are indicated. (G) Localization of Aalpi-18529 in the ovaries of adult females. The nucleic acid probe targeting Aalpi-18529 was conjugated to the dual fluorophore Cy3 (red). Probes targeting a scrambled nucleotide sequence were used as the control for piRNA. Egg chambers were stained with DAPI to visualize nurse cell nuclear morphology. BC, blank control; NC, negative control. The images were acquired using an All-in-One Fluorescence microimaging system BZ-X800/BZ-X810 (KEYENCE).

Article Snippet: Ovaries were mounted in Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Cat#P0131) and imaged 3– 10 days after mounting.

Techniques: Expressing, Marker, Sequencing, Control, Staining, Negative Control, Fluorescence

Journal: bioRxiv

Article Title: AalpiRNA-18529 regulates vitellogenesis of Aedes albopictus via the Gadd45a -mediated JNK-dependent nurse cell apoptosis pathway

doi: 10.1101/2024.08.20.608798

Figure Lengend Snippet: (A) Ovaries from treatment and control groups were stained with TUNEL assay for apoptotic cells (green), and nuclei were stained with DAPI (blue). Representative pictures of ovaries dissected at 24 h PMB are shown. The right insets show a zoomed image of a typical TUNEL-positive (purpe frame, blue arrow) or TUNEL-negative chamber (yellow frame) and TUNEL-positive germarium (red frame, red arrow). Scale bar: 100 µm. (B) The percentage of TUNEL-positive nurse cells. A total of 15 chambers were randomly selected for TUNEL-positive nurse cell counts in ovaries per adult female, with 8 females per group. All data are shown as the mean ± SEM. Student’s t- test was used to compare the means between two groups, and one-way ANOVA was used to compare the means among different groups. * P < 0.05. ** P < 0.01. *** P <0.001; ns, no significance.

Article Snippet: Ovaries were mounted in Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Cat#P0131) and imaged 3– 10 days after mounting.

Techniques: Control, Staining, TUNEL Assay