Figure 5. GPR68 expression is detected in endothelial cells of small-diameter vessels.

(A) Murine Gpr68 mRNA expression profile determined by qRT-PCR. Gapdh was used as the reference gene. The expression levels were normalized to spleen. n=2~3. (B) Representative images of colorimetric RNAscope in situ hybridization for Gpr68 vascular endothelial cells of small diameter blood vessels in brain, pancreas and liver. Scale bars: 25 μm. Arrows indicate cells with positive signal. (C) Schematic diagram of the BAC transgenic construct that is integrated in the genome of Gpr68 eGFP reporter mice. The blue A-box is the homologous sequence that guides recombination and insert eGFP after the start codon of Gpr68. Poly-A sequence following the eGFP blocks the transcription of the downstream genes within the construct (including Gpr68). pA: poly-A sequence; AmpR, amplicilin-resistance genes; R6Kγ, origin of DNA replication (AmpR and R6Kγ are used in the initial selection of BAC constructs). (D) FACS plot of primary endothelial cells isolated from the bladder of Gpr68-eGFP reporter mice. Cells that are CD31+ and CD45− are shown, and grouped in the GFP+ and GFP− populations. (E) Normalized RNA levels (RPKM) of Gpr68 and Piezo1 from the RNAseq data of GFP+ and GFP− endothelial cells. Bladder cells from 3 batches of isolation and sorting (10 mice in total) were pooled and subjected to RNAseq. (F) Representative images of GFP antibody staining in arteries of 1^st order (1°), 2^nd order (2°), 3^rd order (3°) superior mesenteric vessels, and the vessels in the wall of small intestine (w). Scale bars: 50 μm for 1° and 2° vessels, 25 μm for 3° vessels, and 10 μm for vessels in the small intestine wall. 8 groups of vessels from 4 mice were examined.