Fig 1.
mRNA level of hORs with or without the TAR-Tat system.
(a) The design of the control plasmid and the +TAR-Tat plasmid were used in this study. The plasmid, which has a CMV promoter and encodes hOR, was used as a control (pBApo-CMV neo vector). With the TAR-Tat system, the TAR sequence is incorporated in the 5ʹ untranslated region and the Tat sequence is downstream of the hOR sequence (pHEK293 Ultra Expression Vector I). (b) The principle of the TAR-Tat system. The Tat, transcribed and expressed with the hOR, binds to the TAR in the untranslated region. Then, the activated TAR phosphorylates RNA polymerase II and results in the promotion of transcription. (c) The mRNA level of OR1A1/6N2/51M1 was measured by real-time RT-PCR and analyzed by ΔCt analysis. Data are expressed as average (n = 3) ± SE (error bars). ***P < 0.005 and ****P < 0.001, unpaired t-test, for comparison of control and with the TAR-Tat system.
Fig 2.
Expression level of hORs with or without the TAR-Tat system.
(a) Principle of the HiBiT cell surface expression assay. Nonlytic detection reagent containing the substrate furimazine and LgBiT protein binds to hORs that express on the cell surface. LgBiT is membrane-impermeable and cannot bind to intracellular hORs. (b) Luminescence signals of OR1A1/6N2/51M1 expressed on the cell surface. Data are expressed as average (n = 4) ± SE (error bars). (c) Principle of the HiBiT expression assay. Lytic reagent disrupts cells, and the substrate furimazine and LgBiT protein can bind to hORs in the cells. (d) Luminescence signals of OR1A1/6N2/51M1 expressed in cell lysates. Data are expressed as average (n = 4) ± SE (error bars). **P < 0.01 and ***P < 0.005, unpaired t-test, for comparison of control and with the TAR-Tat system.
Fig 3.
Dose–response curves of OR1A1 to (+)-carvone with or without the TAR-Tat system.
The responses of OR1A1 against (+)-carvone were evaluated using the control plasmid (OR1A1/pBApo) or the plasmid having the TAR-Tat system (OR1A1/pHEK). As a negative control, pBApo-CMV neo vector and pHEK Ultra Expression Vector I were used for mock cells. The luminescence was normalized to a maximum value of 100. The y-axis denotes the normalized response, and the x-axis represents the concentration of (+)-carvone. Data are expressed as average (n = 3) ± SD (error bars). *P < 0.05 and **P < 0.005, unpaired t-test, for comparison of control and with the TAR-Tat system.
Fig 4.
Screening of n-hexanal receptors from the hOR repertoire with or without the TAR-Tat system.
The responses of 379 hORs against 500 μM n-hexanal were comprehensively measured using the control plasmid (hORs/BApo) (a) or the plasmid with the TAR-Tat system (hORs/pHEK) (b) (n = 1). Those with ΔL >500 or <−500 are shown in black for the control and magenta for the TAR-Tat system, and all others are shown in gray. X-axis bars represent OR families.
Fig 5.
Dose–response curves of hORs to n-hexanal with or without the TAR-Tat system.
The dose-responses of hORs that show responses in the screening against n-hexanal were measured using the control plasmid (hOR/pBApo) or the plasmid with the TAR-Tat system (hOR/pHEK). Luminescence was normalized to a maximum value of 100. The y-axis denotes the normalized response, and the x-axis represents the concentration of (+)-carvone. The fitting curves were shown if R2 were above 0.7. Data are expressed as average (n = 3) ± SD (error bars). *P < 0.05 and **P < 0.005, unpaired t-test, for comparison of control and with the TAR-Tat system.
Fig 6.
Inhibitory response of OR2M3 against n-hexanal.
(a) The inhibitory effects of n-hexanal on the constitutive activity of OR2M3. Luminescence was normalized to a minimum value of −100. (b) Dose–response curves of OR2M3 to 3-mercapto-2-methyl-1-pentanol using OR2M3/pHEK plasmid. Luminescence was normalized to a maximum value of 100. (c) Dose–response curves of the inhibitory response of OR2M3 against n-hexanal in the presence of agonist (500 nM 3-mercapto-2-methyl-1-pentanol) using OR2M3/pHEK plasmid. 100 indicate the maximum response value of OR2M3 for 3-mercapto-2-methyl-1-pentanol. Data are expressed as average (n = 3) ± SD (error bars). *P < 0.05 and **P < 0.005, unpaired t-test, for comparison of control and with the TAR-Tat system.