Additional file 8: Figure S6. of Detection of pup odors by non-canonical adult vomeronasal neurons expressing an odorant receptor gene is influenced by sex and parenting status

Investigation of activation of Olfr692-expressing cells by aliphatic acids in vivo and odorant screening in Olfr692-expressing heterologous cells using SEAP assay. (a) Olfr692-expressing cells are not activated by short-chain aliphatic acids, such as heptanoic, octanoic, nonanoic, and decanoic acids. See also Additional file 7: Table S1 and references [49, 50]. (b) Screening of Olfr692-expressing artificially modified Hana3a heterologous cells with aliphatic acids presented at different concentrations shows no stimulus-induced activity (alkaline phosphatase output in black lines; positive control response for receptor Olfr544 [49] in red line). Responses were normalized against maximum response in Olfr544-expressing control cells exposed to nonanedioic acid. Mean ± SEM. lu, VNO lumen. Scale bars represent 100 μm. Nuclear staining is To-Pro-3 labeling (blue). (c) Functional assay with a panel of 70 odorants in Olfr692-expressing Hana3a cells. Although serial dilutions in half-log steps from 10–3–10–7 M were used, only the concentrations which gave the maximum fold-change for Olfr692 are shown for each odorant. The x-axis indicates the SEAP signal fold-change for Olfr692-expressing cells relative to Hana3a cells with no OR. The y-axis shows signal fold-change over baseline. Stimulus categories are color-coded, to indicate stimuli that activate VSNs in vitro, pheromones and kairomones, stimuli that can still be detected by mice even in the absence of a functional MOE (AC3-/- mice), or known class I OR ligands. (c’) Immunocytochemistry against the Rho tag to show cell surface expression of Olfr692 (red) in Hana3a transfected cells. (d) Full concentration-response curves for odorants which gave a fold-change over baseline ≥2.5 and fold-change over no-OR control >1.0 in (c). P values are for interaction of receptor and concentration of odorant, two-way ANOVA. Colored lines indicate Hana3a cells expressing Olfr692, in comparison with "no-OR" control cells (black lines). (PDF 1.97 MB)