Fig 2: The expression of TRPV1 was increased in the CYP group, and TRPV1 colocalized with GPR18 in the L6-S1 DRGs 24 h after intraperitoneal injection of saline or CYP. (A) Analysis of the mRNA expression of TRPV1 in the bladders (n=6). (B and C) Analysis of the protein expression of TRPV1 and representative Western blotting image of TRPV1 in the bladders (n=6). (D) The colocalization of TRPV1 and GPR18 in the L6-S1 DRGs. Student's t-test was used for statistical analysis. *P<0.05; ***P<0.001.

Fig 3: The expression of GPR18 was decreased in the bladders and DRGs of rats with CYP-induced cystitis 4, 24, and 48 h after intraperitoneal injection of saline or CYP. (A) Analysis of the mRNA expression of GPR18 in the bladders (n=6). (B and C) Analysis of the protein expression of GPR18 and representative Western blotting images of GPR18 in the bladder (n=6). (D) Analysis of the coexpression of GPR18 (red) and PGP9.5 (green) in normal bladder sections using immunofluorescence. (E) Analysis of the mRNA expression of GPR18 in the L6-S1 DRGs (n=6). (F and G) Analysis of the expression and distribution of GPR18 in the L6-S1 DRGs using immunofluorescence (n=6). The arrows indicate the afferent nerves that supply the bladder. Student's t-test was used for statistical analysis. *P<0.05; **P<0.01; ***P<0.001; ns, no significance.

Fig 4: GPR18 is a target gene of the transcription factor ETS2. (A) mRNA expression levels of GPR18 were determined following the transfection of H9c2 (2-1) cells with an ETS2 overexpressing plasmid. (B) mRNA expression levels of GPR18 were determined following the transfection of H9c2 (2-1) cells with si-ETS2. (C) Protein expression levels of GPR18 following the transfection of cells with either ETS2 overexpression plasmid or si-ETS2. ***P<0.001 vs. control group. GPR18, G-protein coupled receptor 18; si, small interfering RNA; ETS2, E26 transformation-specific transcription factor 2; NC, negative control.

Fig 5: miR-155 regulates cell viability and apoptosis through targeting GPR18 in H9c2 (2-1) cells. (A) mRNA expression levels of GPR18 were determined using reverse transcription-quantitative PCR following the overexpression and silencing of miR-155 using a mimic and inhibitor, respectively in H9c2 (2-1) cells. (B) Protein expression levels of GPR18 were determined using western blotting following the overexpression and silencing of miR-155 using a mimic and inhibitor, respectively in H9c2 (2-1) cells. (C) Cell viability of H9c2 (2-1) cells. (D) Semi-quantification of apoptosis in H9c2 (2-1) cells. (E) Apoptosis in H9c2 (2-1) cells. **P<0.01 and ***P<0.001 vs. control group. NC, negative control; miR, microRNA; GPR18, G-protein coupled receptor 18.