Processed at 55uC for 30 min in the stripping buffer.Serial Dilution Assays on Plate Media and BuffersIn this work, the following growth media were used: YPD (10 g/ liter yeast extract, 20 g/liter tryptone, and 20 g/liter glucose), SC (6.7 g/liter yeast nitrogen base, 20 g/liter glucose, and the appropriate yeast synthetic dropout medium supplements). NaCl, KCl, LiCl and sorbitol were added at various concentrations as indicated. Solid culture was performed on 1.5 agar plates of YPD or YPD with NaCl, KCl, LiCl and sorbitol at the indicated concentration. Stripping buffer contains 62.5 mM Tris-HCl (pH 6.8), 2 SDS and 0.1 M 2-Mecaptoethanol. To estimate the contribution of activation of the HOG pathway, it was necessary to complement the western blot assay with the qualitative plate growth assay. For the dilution assays, cells in exponential phase at a concentration of 16106 cells/ml were diluted 10-fold a total of four times, and 5 ml of each dilution, including the starting dilution of 16106 cells/ml, was plated on YPD plates containing various stressinducing agents.Latrunculin TreatmentCells in exponential phase (OD600,0.8?.0) were treated with 100 mM latrunculin B (Lat B, Invitrogen; from stock solution 10 mM in ethanol) for 20 min. The same volume of ethanol was added to the control cell culture.Western BlottingCells (5 ml of culture; OD600 = 0.6) were taken at the indicated time points, and collected by brief centrifugation. Dual phosphorTable 1. Strains used in this study.Strain BY4741 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 HGenotype MATa his3D1 leu2D0 met15D0 ura3D0 MATa his3D1 leu2D0 met15D0 ura3D0 hog1D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 pbs21D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk1D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 EPZ015666 ssk22D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk1D::natMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::natMX ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk22D::natMX ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::natMX ssk22D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk2D::natMX ssk1D:: HIS3 MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk2D::natMX ssk1D:: HIS3 MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk22D::natMX ssk1D:: HISSource INVITROGEN INVITROGEN INVITROGEN INVITROGEN 15857111 INVITROGEN INVITROGEN INVITROGEN This study This study This study This study This study This study This studydoi:10.1371/journal.pone.0054867.tAlternative Activation of Ssk2p in Osmotic StressMicroscopy and Rhodamine-Phalloidin StainingRhodamine-phalloidin (Tazemetostat Rd-phalloidin) staining of actin in yeast was performed as described [30]. Cells were viewed with a laser scanning confocal microscope (FV1000, OLYMPUS; Zeiss 510). In addition, images were displayed using FLV-ASW.Results Hog1p can be Phosphorylated and Activated in the ssk1Dste11D Double MutantIn the HOG pathway, Ssk1p is considered as the activator of Ssk2p and Ssk22p [20]. Early epistasis analysis placed Ssk2p and Ssk22p upstream of Pbs2p and downstream of Ssk1p [5,8]. If Ssk1p is the sole activator of the Ssk2p and Ssk22p, the double mutant ssk1Dste11D should be as osmosensitive as pbs2D and hog1D mutants and fail to phosphorylate Hog1p upon osmotic shock. However, some studies have found that expression of most osmoregulated genes are clearly induced or repressed in ssk1Dste11D mutant under severe osmotic stress (0.5 M KCL.Processed at 55uC for 30 min in the stripping buffer.Serial Dilution Assays on Plate Media and BuffersIn this work, the following growth media were used: YPD (10 g/ liter yeast extract, 20 g/liter tryptone, and 20 g/liter glucose), SC (6.7 g/liter yeast nitrogen base, 20 g/liter glucose, and the appropriate yeast synthetic dropout medium supplements). NaCl, KCl, LiCl and sorbitol were added at various concentrations as indicated. Solid culture was performed on 1.5 agar plates of YPD or YPD with NaCl, KCl, LiCl and sorbitol at the indicated concentration. Stripping buffer contains 62.5 mM Tris-HCl (pH 6.8), 2 SDS and 0.1 M 2-Mecaptoethanol. To estimate the contribution of activation of the HOG pathway, it was necessary to complement the western blot assay with the qualitative plate growth assay. For the dilution assays, cells in exponential phase at a concentration of 16106 cells/ml were diluted 10-fold a total of four times, and 5 ml of each dilution, including the starting dilution of 16106 cells/ml, was plated on YPD plates containing various stressinducing agents.Latrunculin TreatmentCells in exponential phase (OD600,0.8?.0) were treated with 100 mM latrunculin B (Lat B, Invitrogen; from stock solution 10 mM in ethanol) for 20 min. The same volume of ethanol was added to the control cell culture.Western BlottingCells (5 ml of culture; OD600 = 0.6) were taken at the indicated time points, and collected by brief centrifugation. Dual phosphorTable 1. Strains used in this study.Strain BY4741 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 HGenotype MATa his3D1 leu2D0 met15D0 ura3D0 MATa his3D1 leu2D0 met15D0 ura3D0 hog1D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 pbs21D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk1D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk22D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk1D::natMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::natMX ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk22D::natMX ste11D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ssk2D::natMX ssk22D::KanMX MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk2D::natMX ssk1D:: HIS3 MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk2D::natMX ssk1D:: HIS3 MATa his3D1 leu2D0 met15D0 ura3D0 ste11D::KanMX ssk22D::natMX ssk1D:: HISSource INVITROGEN INVITROGEN INVITROGEN INVITROGEN 15857111 INVITROGEN INVITROGEN INVITROGEN This study This study This study This study This study This study This studydoi:10.1371/journal.pone.0054867.tAlternative Activation of Ssk2p in Osmotic StressMicroscopy and Rhodamine-Phalloidin StainingRhodamine-phalloidin (Rd-phalloidin) staining of actin in yeast was performed as described [30]. Cells were viewed with a laser scanning confocal microscope (FV1000, OLYMPUS; Zeiss 510). In addition, images were displayed using FLV-ASW.Results Hog1p can be Phosphorylated and Activated in the ssk1Dste11D Double MutantIn the HOG pathway, Ssk1p is considered as the activator of Ssk2p and Ssk22p [20]. Early epistasis analysis placed Ssk2p and Ssk22p upstream of Pbs2p and downstream of Ssk1p [5,8]. If Ssk1p is the sole activator of the Ssk2p and Ssk22p, the double mutant ssk1Dste11D should be as osmosensitive as pbs2D and hog1D mutants and fail to phosphorylate Hog1p upon osmotic shock. However, some studies have found that expression of most osmoregulated genes are clearly induced or repressed in ssk1Dste11D mutant under severe osmotic stress (0.5 M KCL.
