Performance of non-specific (expected negative controls) and positive controls for the V2R pharmacoperone assay.

<p>The non-specific compounds were selected from reagents that alter the level of cyclic nucleotides, block Ca2⁺ and Na channels, inhibit calmodulin, inhibit transcription, activate a range of receptors, cross link membrane proteins and otherwise perturb the cell. The dotted lines parallel to the X-axis show the response of the cells in the presence of medium only (i.e., no added drugs). The drugs, used at a concentration of 1 µg/ml were: 1. Urotensin II (neurosecretory peptide); 2. Octreotide Related Peptide; 3. Somatostatin; 4. Bombesin; 5. Calcitonin (salmon); 6. Growth Hormone Releasing Factor; 7. Thyrotropin Releasing Factor; 8. Galanin (human); 9. NPSF-Amide (SLAAPQRF-NH2); 10. Neuromedin U (rat); 11. BI 679 (the growth hormone releasing peptide, hexarelin); 12. Adiponectin (a hormone with broad impact on metabolism); 13. 1-(3-Dimethylaminopropyl)-3-Ethylcarbodiimide (a water-soluble protein crosslinker); 14. O2′-Monosuccinyl Guanosine 3′-5′-Cyclic Monophosphate Tyrosine Methyl Ester (cGMP analog); 15. 1-Ethyl-3-(3-Dimethylamino-Propyl)Carbodiimide-HCl (a water-soluble protein crosslinker); 16. D-β-3,4-dihydroxy-Phenylalanine (D-DOPA); 17. L-Noradrenaline; 18. Trifluoperazine (calmodulin antagonist); 19. Histone (from calf thymus) Type II-S (a basic protein); 20. N6-2′-O-Dibutyryladenosine 3′-5′-Cyclic Monophosphate (a cAMP analog); 21. Spermine; 22. Ouabain Octahydrate (Strophanthin-G) (sodium ion channel antagonist); 23. 3-Hydroxytyramine; 24. p-Nitrophenyl Phosphate; 25. 8-(4-Chlorophenylthio)-Adenosine 3′:5′-Cyclic Monophosphate; 26. Carbonyl Cyanide m-Chlorophenylhydrazone; 27. Guanosin-5′-triphosphate; 28. Adenylyl-imidodiphosphate (AMP-PNP); 29. p-Nitrophenyl-β-D-Galactopyranoside; 30. Cytochrome-C; 31. Concanavalin A (a plant lectin that interacts with plasma membrane glycoproteins); 32. L-1-Tosylamide-2-Phenyl-Ethylchloromethyl Ketone (inhibitor of trypsin-like enzymes); 33. Actinomycin D (transcription inhibitor); 34. β -Nicotinamide Adenine Dinucleotide (β-NAD); 35. Adenosine 5′-Monophosphoric Acid; 36. Nifedipine (Ca2+ ion channel antagonist); 37. D600 (Ca2+ ion channel antagonist); 38. 2-n-Propyl-Amino-indine; 39. Veratrine (Na channel inhibitor); 40. Vinblastine (microtubule inhibitor); 41. Cytochalasin D (blocks cellular actin polymerization); 42. Polyinosinic-Polycytidylic Acid (Poly[I]-Poly[C]) polymer; 43. Forskolin (activator or adenylate cyclase); 44. A23187 (Ca2+ ionophore); 45. SQ23,377 Ionomycin (Ca2+ ionophore); 46. U-73343 (inhibitor of inositol phosphate metabolism); 47. Creatine Kinase; 48. Deferoxamine Mesylate (metal chelator); 49. Flavin Mononucleotide; 50. [-]-Norepinephrine; 51. Adenosine-3,5′-Cyclic Monophosphothioate, Rp-Isomer; 52. N-nitro-L-arginine L-NAME Methyl Ester; 53. Acyline (GnRH peptide antagonist); 54. Asp2-GnRH (GnRH analog that binds GnRHR mutant E⁹⁰K); 55. Buserelin (GnRHR peptide agonist); 56. Control (medium only); 57. In3 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 58. Q89 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 59. TAK-013 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 60. SR121463B (non-specific for the GnRHR assay, non-peptide V2R antagonist).</p

Performance of non-specific (expected negative controls) and positive controls for the GnRHR pharmacoperone assay.

<p>The non-specific compounds were selected from reagents that alter the level of cyclic nucleotides, block Ca2⁺ and Na channels, inhibit calmodulin, inhibit transcription, activate a range of receptors, cross link membrane proteins and otherwise perturb the cell. The dotted lines parallel to the X-axis show the response of the cells in the presence of medium only (i.e., no added drugs). The drugs, used at a concentration of 1 µg/ml were: 1. Urotensin II (neurosecretory peptide); 2. Octreotide Related Peptide; 3. Somatostatin; 4. Bombesin; 5. Calcitonin (salmon); 6. Growth Hormone Releasing Factor; 7. Thyrotropin Releasing Factor; 8. Galanin (human); 9. NPSF-Amide (SLAAPQRF-NH2); 10. Neuromedin U (rat); 11. BI 679 (the growth hormone releasing peptide, hexarelin); 12. Adiponectin (a hormone with broad impact on metabolism); 13. 1-(3-Dimethylaminopropyl)-3-Ethylcarbodiimide (a water-soluble protein crosslinker); 14. O2′-Monosuccinyl Guanosine 3′-5′-Cyclic Monophosphate Tyrosine Methyl Ester (cGMP analog); 15. 1-Ethyl-3-(3-Dimethylamino-Propyl)Carbodiimide-HCl (a water-soluble protein crosslinker); 16. D-β-3,4-dihydroxy-Phenylalanine (D-DOPA); 17. L-Noradrenaline; 18. Trifluoperazine (calmodulin antagonist); 19. Histone (from calf thymus) Type II-S (a basic protein); 20. N6-2′-O-Dibutyryladenosine 3′-5′-Cyclic Monophosphate (a cAMP analog); 21. Spermine; 22. Ouabain Octahydrate (Strophanthin-G) (sodium ion channel antagonist); 23. 3-Hydroxytyramine; 24. p-Nitrophenyl Phosphate; 25. 8-(4-Chlorophenylthio)-Adenosine 3′:5′-Cyclic Monophosphate; 26. Carbonyl Cyanide m-Chlorophenylhydrazone; 27. Guanosin-5′-triphosphate; 28. Adenylyl-imidodiphosphate (AMP-PNP); 29. p-Nitrophenyl-β-D-Galactopyranoside; 30. Cytochrome-C; 31. Concanavalin A (a plant lectin that interacts with plasma membrane glycoproteins); 32. L-1-Tosylamide-2-Phenyl-Ethylchloromethyl Ketone (inhibitor of trypsin-like enzymes); 33. Actinomycin D (transcription inhibitor); 34. β -Nicotinamide Adenine Dinucleotide (β-NAD); 35. Adenosine 5′-Monophosphoric Acid; 36. Nifedipine (Ca2+ ion channel antagonist); 37. D600 (Ca2+ ion channel antagonist); 38. 2-n-Propyl-Amino-indine; 39. Veratrine (Na channel inhibitor); 40. Vinblastine (microtubule inhibitor); 41. Cytochalasin D (blocks cellular actin polymerization); 42. Polyinosinic-Polycytidylic Acid (Poly[I]-Poly[C]) polymer; 43. Forskolin (activator or adenylate cyclase); 44. A23187 (Ca2+ ionophore); 45. SQ23,377 Ionomycin (Ca2+ ionophore); 46. U-73343 (inhibitor of inositol phosphate metabolism); 47. Creatine Kinase; 48. Deferoxamine Mesylate (metal chelator); 49. Flavin Mononucleotide; 50. [-]-Norepinephrine; 51. Adenosine-3,5′-Cyclic Monophosphothioate, Rp-Isomer; 52. N-nitro-L-arginine L-NAME Methyl Ester; 53. Acyline (GnRH peptide antagonist); 54. Asp2-GnRH (GnRH analog that binds GnRHR mutant E⁹⁰K); 55. Buserelin (GnRHR peptide agonist); 56. Control (medium only); 57. In3 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 58. Q89 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 59. TAK-013 (non-specific for the V2R assay; non-peptide GnRH antagonist, structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022784#pone.0022784-Conn4" target="_blank">[28]</a>); 60. SR121463B (non-specific for the GnRHR assay, non-peptide V2R antagonist).</p

Descriptive Statistics of Signals by Concentrations (V2R).

<p>Descriptive Statistics of Signals by Concentrations (V2R).</p

Mean profiles of signals by day and plate for the V2R pharmacoperone assay.

<p>Line plots were performed to reveal patterns of date by date variations, and plate to plate systematic variations.</p

Descriptive Statistics of Signals by Concentrations (GnRHR).

<p>Descriptive Statistics of Signals by Concentrations (GnRHR).</p

Spatial uniformity assessment of the GnRHR pharmacoperone assay.

<p>The signals are plotted against the well number. The wells are ordered by the rows and columns in order to explore any patterns of the edge, drift effect and other systematic source of variability.</p

A residual plot for the GnRHR pharmacoperone assay.

<p>To assess the validity of the model assumptions, the plot of the Studentized residuals versus the predicted values, along with 95% confidence interval, is displayed.</p

A scatterplot with regression fit for the GnRHR pharmacoperone assay.

<p>A linear regression analysis was attempted to evaluate the linearity/association of the dose-response of pharmacoperone and concentrations of dose by estimating a polynomial function.</p

Linearity assessment for the V2R pharmacoperone assay.

<p>Using a pharmacoperone drug, the relation between the dose response and dose concentrations was evaluated.</p

Linear regression parameter estimates (GnRHR).

<p>Linear regression parameter estimates (GnRHR).</p