17 cells [3,25,26]. Interestingly, antagonizing D1 receptor function inhibits the differentiation of Th-17 cells [25]. Recent studies have shown that functional D1 dopamine receptors are expressed in dendritic cells and naive T-cells and the specific activation of these D1 receptors by dopamine promotes the differentiation of the T- cells into Th-2 and Th-17 cells [25–27]. An earlier study showed that D1 dopamine receptors suppress the differentiation of na¨ıve T-cells into regulatory T-cells (T-regs) cells that are involved in the immune suppression response [28]. Our observations suggest that miR-142-3p mediated regulation of D1 receptor expression might contribute to T-cell differentiation. While miR-142-3p function has been primarily described in the hematopoietic and immune systems, our results suggest that this microRNA is also expressed in the brain and in the neuronal CAD cell line. These results are consistent with a previous study which showed that mir-142-3p is not only expressed in the brain but is enriched in purified striatal post-synaptic densities along with the Argonaute protein-2, which is a key protein involved in microRNA-mediated post-transcriptional regulation [29]. This suggests that miR-142-3p-mediated post-transcriptional regulation might regulate translation of D1 receptor protein in dendritic spines. This novel molecular mechanism might dynamically regulate D1 receptor expression in the dendritic spines in response to neuronal excitation. Our novel finding that miR-142-3p is expressed in the brain and directly regulates the expression of D1 dopamine receptor protein suggests that miR-142-3p might play an important role in modulating D1 receptor-dependent behav- iors. Supporting Information Figure S1 Schematic representation of the binding sites for miR-294/295-3p, miR-142-3p and miR-10a* in D1 receptor 39UTR and the seed recognition nucleotides that were targeted for mutation. Partial nucleotide sequence (from nucleotides 560 to 1250) of the 1277 bp D1 39UTR that shows the putative binding sites (highlighted in yellow) for miR- 294/295-3p, miR-142-3p and miR-10a*. The poly A signal site is indicated in bold letters. The top strand represents the wild type sequence and the bottom strand is the mutated version. The specific nucleotides that were mutated within each seed recogni- tion sequence (underlined) are represented by a dash. Conserved nucleotides are represented by a *. (TIF) Figure S2 Sensitivity and selectivity of molecular probes used to detect D1 dopamine receptor in this study. (A) Raw traces from a representative real-time PCR detected using D1 receptor-specific TaqManH probe shows the sensitivity of the amplification method to identify D1 receptor expression in serially diluted total mouse brain cDNA samples. (B) Cumulative data showing a linear correlation between the Ct values (cycle numbers) and concentration of serially diluted total mouse brain cDNA that were amplified and detected using TaqManH probes specific for D1 dopamine receptor. The data points represent mean (6 SEM) of 4 independent RT-PCR reactions and were fitted with linear regression. (C) Representative western blot showing the level of mouse D1 receptor protein in AtT-20 neuroendocrine cells (which does not express endogenous D1 receptor) transiently-transfected with empty plasmid vector (lane 1) or a constitutive expression plasmid encoding the mouse D1 dopamine receptor (lane 2). Each lane was loaded with 30 mg of total cell protein and the D1 receptor protein detected using the anti-D1 receptor rat monoclonal antibody. Multiple glycosylated forms of mature D1 receptor are detected with this antibody. (D) D1 Receptor Expression Is Regulated by MicroRNA Representative western blot showing relative expression level of endogenous D1 receptor protein in non-differentiated CAD cells and mouse striatum. We loaded 100 mg of total protein from CAD cells in lane 1 and 1, 5, 10 and 30 mg of serially diluted total protein from the striatum of a mouse that was one month old in lanes 2 through 5. Two different film exposures as well as the amido black stained blot indicating total protein loaded in each lane is shown. (TIF) Figure S3 D1 receptor post transcriptional regulation is mediated by the 1277 bp region proximal to the stop codon. (A) Schematic representation of the two different D1 receptor 39UTRs (1277 bp and 1684 bp) with the relative location of the two polyadenylation signals at positions 714 and 1635. (B) The two different D1 39UTR regions were sub cloned into a construct with the EGFP reporter gene regulated by the 6400 bp D1 receptor promoter. The D1 receptor 39UT regions replaced the heterologous SV40 39UTR in the recombinant constructs. The three constructs were separately transfected into non-differentiated CAD cells along with a transfection control construct encoding the BAP-FlagTM gene. The inset shows equal EGFP reporter mRNA from all three constructs using real time RT-PCR. In contrast, western blot analysis with EGFP monoclonal antibody shows that EGFP reporter protein was significantly reduced in cells transfected with both D1 receptor 39UTR constructs. The bars represent the mean values 6 s.e.m. (n = 3). *P,0.001, One Way ANOVA, post hoc SNK test. (TIF) Figure S4 The steady-state levels of b-galactosidase reporter mRNA from the D1 receptor 39UTR constructs. Constructs with deletions (A) or mutations in the microRNA binding sites (B) is not significantly different. The various reporter constructs were transiently transfected into non-differentiated CAD cells along with a control BAP-FlagTM construct for monitoring transfection efficiency. The mRNA levels were assayed using real-time RT-PCR with the SYBRH green method and primers specific for b-galactosidase and BAP-FlagTM. The reactions were stopped in the exponential phase and the products run out on an ethidium bromide stained agarose gel. (TIF) Figure S5 The steady-state levels of b-galactosidase reporter mRNA from the D1 receptor 39UTR deletion constructs. Cumulative results comparing b-galactosidase mRNA levels between non-differentiated CAD cells transfected with full length D1 39UTR [pCMV b-gal+D1 39UTR (1277 bp)] or D1 39UTR constructs with deletions [pCMV b-gal+D1 39UTR (526 bp) and pCMV b-gal+D1 39UTR (94 bp)]. Constructs with deletions were not significantly different. The various reporter constructs were transiently transfected into non-differentiated CAD cells along with a control BAP-FlagTM construct for monitoring transfection efficiency. The mRNA levels were assayed using real-time RT-PCR with the SYBRH green method and primers specific for b-galactosidase and BAP-FlagTM. The reactions were stopped in the exponential phase and the products run out on an ethidium bromide stained agarose gel (representa- tive gel is shown in the inset). The bars represent the mean values 6 s.e.m. (n = 3). Not significant, P.0.05, One Way ANOVA. (TIF) PLOS ONE | www.plosone.org 14 November 2012 | Volume 7 | Issue 11 | e49288