DEGs in Conv versus GF within renal clusters

Using data from Ransick, et al (17), table lists differentially expressed genes (DEGs) assigned to each renal cell type in female (Table S1) and male (Table S2) mice.   </p

The Lucy tag is a cleavable signal peptide.

<p>HA-Flag-Rho-Olfr691 (A and B) or HA-Lucy-Flag-Rho-Olfr691 (C and D) constructs were expressed in HEK293T cells along with RTP1S. Cells were fixed and stained with both an HA and Flag antibody (A and C) to detect total tagged OR or surface labeled with the HA and Flag antibodies (B and D) to detect surface-associated OR. HA surface stain is observed only in the absence of the Lucy tag, indicating a functional Lucy cleavage site.</p

Summary of the tissue expression profile of all the novel sensory receptors identified in the mouse whole kidney cDNA.

<p>A ‘+’ sign indicates expression of the corresponding receptor in our RT-PCR screen whereas a ‘−’ sign indicates absence in that particular tissue. All ‘+’ signs in the table were confirmed by sequencing to confirm identity. In each case, the mock sample without reverse transcriptase during cDNA synthesis was negative.</p><p>Summary of the tissue expression profile of all the novel sensory receptors identified in the mouse whole kidney cDNA.</p

RT-PCR with mouse whole kidney cDNA as template to identify novel renal taste receptors.

<p>Tas2r108 (A), Tas2r119 (B), Tas2r135 (C), Tas2r137 (D), Tas2r138 (E), Tas2r140 (F), Tas2r143 (G), Tas1r1 (H), Tas1r2 (I) and Tas1r3 (J) PKD1L3 (K) and G_NAT3 (L) expression detected in the mouse whole kidney cDNA by RT-PCR and confirmed by sequencing. Mock controls without RT are negative in all the lanes. The white arrow indicates the band of the expected size for each olfactory receptor.</p

RT-PCR with mouse whole kidney cDNA as template to identify novel renal olfactory receptors.

<p>Olfr99 (A), Olfr545 (B), Olfr691 (C), Olfr693 (D), Olfr31(E) and Olfr1426(F) expression is detectible in mouse whole kidney cDNA by PCR and sequencing confirms the identity of amplified products. Mock RT template controls are negative for OR GSP sets and β-actin (not shown). The white arrow indicates the band of the expected size for each olfactory receptor.</p

Conventional RT-PCR confirms expression of the six most highly expressed GPRs and top 25 highly expressed transcripts from the TaqMan screen.

<p>(<b>A</b>) Gprc5c, Gpr56, Gpr116, Gpr137, Gpr48 and Gpr137b were identified as the top six highly expressed GPRs in the mouse kidney based on the TaqMan screen. Mock lanes without RT are negative for all GPRs. The white arrow indicates the expected sized band for each GPR. (<b>B</b>) Whole kidney RT and mock RT reaction mixture were screened to validate expression of top 25 highly expressed targets identified from our Taqman array screen: (1)Actb (2) Gapdh (3) Ppia (4) Pgk1 (5) Ubc (6) Calm1 (7) B2 m (8) Pth1r (9) Ywhaz (10) Calm2 (11) Gpr137b (12) Tm7sf3 (14) Agtr1a (15) Sfrp1 (17) Ptger3 (18)Tfrc (19) Hprt (22) Fzd4 (23) Avpr2 (25) Polr2a. All products were sequenced to confirm their identities. The white arrow indicates the expected size band for each receptor.</p

The Lucy tag does not alter OR signaling.

<p>(A) A luciferase reporter assay was performed for both Rho-Olfr691(R-691) and Lucy-Rho-Olfr691 (L-R-691) with and without RTP1S. Cells expressing the Olfr691 constructs were grown in a 96-well plate and exposed to the known Olfr691 ligand, isovaleric acid (0-5 mM). Error bars represent the SEM. By ANOVA and Student-Newman-Keuls, no concentrations of isovaleric acid activated R-691 (n/s = no significance). For R-691 + RTP1S, L-691, and L-691 + RTP1S, P ≤ 0.05 for 0 mM vs. all doses of isovaleric acid (marked by an *). In addition, for R-691 + RTP1S, P ≤ 0.05 for 5 vs 0.5, 0.25 and 0.1, 1 vs 0.25 and 0.1, 0.5 vs 0.1. For L-691, P ≤ 0.05 for 5 vs 0.1, 0.25 and 0.5. For L-691 + RTP1S, P ≤ 0.05 for 5 vs 0.5, 0.25 and 0.1, 1 vs 0.5, 0.25 and 0.1, 0.5 vs 0.1. (B) A luciferase reporter assay was performed for mOREG (EG), Rho-mOREG (R-EG), Lucy-mOREG (L-EG) and Lucy-Rho-mOREG (L-R-EG), all in the absence of RTP1S. Cells expressing the mOREG constructs were grown in a 96-well plate and exposed to the known mOREG ligand, eugenol (100-300 µM). Error bars represent the SEM. By ANOVA and Student-Newman-Keuls, all concentrations of eugenol significantly activated (P ≤ 0.05) eg, R-EG, L-EG and L-R-EG as compared to 0 µM (marked by an *). In addition, 100 and 300 µM eugenol were significant from each other (P ≤ 0.05) for both L-EG and L-R-EG. In both A and B, the Firefly: Renilla ratio was measured and compared to the non-treated control. An increase in the ratio indicates OR activation. Both Lucy-Rho-691 and Lucy-tagged mOREG constructs were activated with their ligands indicating that the Lucy tag does not alter OR signaling.</p

Immunohistochemistry showing surface expression of ORs.

<p>Each OR is shown under the experimentally determined condition which allowed for optimized surface expression in HEK293T cells. The surface trafficking conditions vary for each OR and we have published the corresponding conditions for Olfr99, 545, 691 and 693 previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone.0111053-Shepard1" target="_blank">[25]</a>. Briefly, Olfr31 requires co-expression of RTP1S; Olfr691 & Olfr693 require presence of N-terminal Lucy tag along with co-expression of RTP1S; Olfr99, Olfr545 & Olfr1426 requires presence of N-terminal Lucy tag along with co-expression of RTP1S and Ric8b respectively. Olfr31 requires co-expression of RTP1S and Olfr1426 failed to reach the membrane surface at all the tested conditions. HEK293T cells were first stained with a poly-flag antibody (surface) then subsequently permeabilized and stained with a mono-flag antibody (total). The images were taken at equal exposure between all surface and total conditions. Surface images are marked with either a+or – in their lower right-hand corners to indicate the presence or absence of surface expression, respectively. Images in (B) have been enhanced to better display surface expression. Images in (A) are presented as they were taken. Unenhanced images for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone-0111053-g004" target="_blank">Figure 4B</a> can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone.0111053.s001" target="_blank">Figure S1</a>.</p

Ligand screening for Olfr691.

<p>Olfr691 responds to published short chain fatty acids, isovalerate and valerate, in a dose dependent manner when co-expressed with RTP1S (A). Further ligand screening shows that Olfr691 responds to wide range of saturated short and medium chained fatty acids, from propionate to octanoate, but not including formate and acetate (B). NT represents measurements obtained from non-treated cells (with no ligand) transfected with Olfr691 and RTP1S.</p

The Lucy tag works synergistically with accessory proteins and tags to promote surface expression of ORs.

<p>The Lucy tag works synergistically with accessory proteins and tags to promote surface expression of ORs.</p