Figure 3

DRD2 SNP cluster rs6276, rs6277 decreases D2R expression, modulates β-catenin phosphorylation and increases cell proliferation in vitro. (a) Schema of the relative positions of the SNPs rs6276 and rs6277 in the human DRD2 gene. (b) The joint presence of SNPs rs6276 and 6277 in human renal proximal tubule cells reduced D2R mRNA levels by 50% (left panel) along with a concomitant 50% decrease in D2R protein levels (middle panel). Levels of phospho-β-catenin (P-β-catenin) also decreased by 40%, relative to control cells without these SNPs (right panel). Insets show representative immunoblots. (c) Transfection of the DRD2 SNP-containing cells with a human DRD2 plasmid without the SNPs significantly boosted D2R mRNA levels 30-fold (left panel), as well as D2R protein levels 2.2-fold (middle panel). Increasing wild-type D2R expression in these cells also increased P-β-catenin levels (right panel, inset showing western blot), relative to the empty vector control. (d) Presence of DRD2 SNPs rs6276 and rs6277 led to a 2.7-fold increase in cellular proliferation as indicated by the Ki-67 marker. All data are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. **P < 0.01, ***P < 0.001; n = 3–4/group; Student’s t-test.