Figure 1.
Viability of yeast cells measured by colony formation and MTT assay after plasma treatment.
(A) Relative percentage of colony formations by yeast cells in water, saline, and YPD after treatment with plasma, compared to Ar gas only. Relative colony forming units (CFU) are calculated as follows: relative CFU (%) = (CFU number of plasma treated yeast/CFU number of Ar gas treated yeast)×100. All value are the average of three technical replicates. (B) The relative level of formazan product measured as absorbance at 490 nm in cells treated with plasma in water, saline, and YPD, compared to that in Ar gas treated cells. Yeast cells were treated with plasma and Ar gas for 3 min. Relative absorbance was calculated as relative absorbance (%) = (absorbance at 490 nm in plasma treated cells/absorbance at 490 nm in Ar gas treated cells)×100. Each value is the average of three technical replicates.
Figure 2.
Morphology and internal structure of yeast cells after plasma treatment.
(A) Cells analyzed by scanning electron microscopy. (B) Cells analyzed by transmission electron microscopy. Yeast cells in water, saline, and YPD were exposed to Ar gas and plasma for 3 min and used for the microscopic analysis.
Figure 3.
Lipid peroxidation and genomic DNA stability analyses.
Yeast cells (2×107) were exposed to Ar gas and plasma for 3 min and used for the analyses of lipid peroxidation and genomic DNA. (A) Production of 4-hydroxynonenal (4-HNE) measured as fluorescence intensity at 500/520 nm in cells treated with plasma in water, saline, and YPD. Each value is the average of three technical replicates. (B) Fluorescence image of cells after Ar gas and plasma treatment in water, saline, and YPD. (C) Agarose gel electrophoresis of genomic DNA. Same amount of genomic DNA (2 µg) was loaded on the 1% agarose gel.
Figure 4.
Phosphorylation of HOG1 MAP kinase after Ar gas and plasma treatment analyzed by Western blot.
The levels of Hog1p expression and phosphorylation are shown in the first and second panel, respectively. The third panel shows the Western blot for actin as loading control. Total protein was extracted from the cells treated with Ar gas and plasma in water, saline, and YPD for 3 min, and run on a 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel.
Figure 5.
pH change after plasma treatment and effects of osmotic strength of background solutions.
(A) pH of water, saline, PBS, and YPD after plasma treatment (left panel) and the effect of PBS on yeast cell viability (right panel). For pH measurements, 1 ml of each solution was exposed to plasma for the indicated time. The effect of PBS was assessed by measuring relative viability of plasma-treated cells compared to that of Ar gas-treated cells. Cells were treated with plasma for 3 min. (B) Viability of yeast cells in different concentrations of NaCl (left panel) and sorbitol (right panel) after plasma treatment. Yeast cells were treated with Ar gas and plasma for 1 and 3 min. (C) Changes in pH of NaCl (left panel) and sorbitol (right panel) solutions during plasma treatment. One ml of saline and sorbitol was exposed to plasma at the indicated concentrations for 10, 30, 60, and 180 sec. In (A) and (B), cell viability was assessed by relative ratio in colony forming units (CFU) calculated as follows: relative CFU (%) = (CFU number of plasma treated yeast/CFU number of Ar gas treated yeast)×100. All values are the average of three technical replicates.
Figure 6.
The level of reactive oxygen species (ROS) and reactive nitrogen species (RNS).
ROS and RNS levels were measured on the surface of solutions (A), and inside solutions (B) and cells (C) after Ar gas and plasma treatments. Absorbance values in (A) were measured by optimal emission spectroscopy and are the average of five replicates. In (B) and (C), all values are normalized, in which the level measured after Ar gas treatment was subtracted from that measured after plasma treatment. Each value is the average of three technical replicates.
Figure 7.
Intracellular level of NO during plasma exposure time and in different concentrations of NaCl.
(A) Intracellular NO level was measured after plasma exposure in saline for 30, 60, and 180 sec. Fluorescence values are normalized, in which the level measured after Ar gas treatment was subtracted from that measured after plasma treatment. (B) NO level was measured in cells incubated in different concentrations of NaCl solution for 5 and 10 min. Each value is the average of three technical replicates.