Id 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control group. doi:10.1371/journal.pone.0053616.gfungal mycelium cultured on regular PDA for 1.5 month only gave a maximum of 244.9 and 1857.2 mg/100 mg DW for GA 24 and total GAs, respectively (Figure 4). These results indicate that aspirin treatment is a powerful approach to triggering GA MedChemExpress Indolactam V production over a short time.Aspirin induced apoptosis in the fungal cells of G. lucidumFungal apoptosis displays certain characters; these include nuclear morphology changes and double-stranded DNA degradation. The latter can be examined by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling (TUNEL) MedChemExpress Oltipraz assays. Apoptosis of aspirin-treated fungal cells was evaluated by TUNEL assay and nuclear staining (Figure 5). Normal fungal cell did not show any fluorescent signal after TUNEL staining, indicating that 18325633 the genomic DNA of normal cells was intact. To evaluate the nuclear morphology changes, 49, 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. In addition, as a control, the chromosomal DNA of normal cells was pre-digested with DNase I and then assessed by TUNEL reaction mixture to show the nuclear morphology. Figure 5 showed nuclei observed in normal fungal cells. Fungal cells treated with 2 mM aspirin showed a few TUNEL positive cells and condensed nuclei were observed when the cells under the same condition were stained byEffect of fungal culture age on ganoderic acids inductionTo produce a maximum amount of GAs, fungal mycelium from different culture stages was tested for GA production after induction with aspirin. Fungal mycelium was cultured on PDA for 4 to 12 days and then was incubated with 4 mM aspirin for 1 day. As shown in Figure 3B and 3C, GA production by the control culture increased with the cultivation time. Furthermore, GA 24 production and total GA production were both significantly enhanced by aspirin across the various ages of fungal mycelium. Maximal GA24 production and total GA production were 515.2 and 5385 mg/100 mg dry weight (DW), respectively, and this was obtained using the 12-days old mycelium. This is a 2.7-fold and 2.8-fold increase compared with the control. In comparison,Enhanced GA Production by Apoptosis in G. lucidumFigure 2. Time course of ganoderic acids and fungal biomass production of Ganoderma lucidum incubated with aspirin. Fungal mycelium was cultured on PDA for 4 days and then incubated with 4 mM aspirin for additional 6 to 48 hr. Fungal biomass (A), accumulation of lanosta-7,9(11), 24-trien-3a-o1-26-oic acid (ganoderic acid 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control group. doi:10.1371/journal.pone.0053616.gFigure 3. Effect of fungal culture age on ganoderic acids induction by aspirin in Ganoderma lucidum. Fungal mycelium cultured on PDA for 4?2 days was incubated with 4 mM aspirin for additional 1 day. Fungal biomass (A), lanosta-7,9(11), 24-trien-3a-o1-26oic acid (ganoderic acid 24) production (B) and total ganoderic acids (total GAs) production (C) by Ganoderma lucidum were determined. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control g.Id 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control group. doi:10.1371/journal.pone.0053616.gfungal mycelium cultured on regular PDA for 1.5 month only gave a maximum of 244.9 and 1857.2 mg/100 mg DW for GA 24 and total GAs, respectively (Figure 4). These results indicate that aspirin treatment is a powerful approach to triggering GA production over a short time.Aspirin induced apoptosis in the fungal cells of G. lucidumFungal apoptosis displays certain characters; these include nuclear morphology changes and double-stranded DNA degradation. The latter can be examined by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling (TUNEL) assays. Apoptosis of aspirin-treated fungal cells was evaluated by TUNEL assay and nuclear staining (Figure 5). Normal fungal cell did not show any fluorescent signal after TUNEL staining, indicating that 18325633 the genomic DNA of normal cells was intact. To evaluate the nuclear morphology changes, 49, 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. In addition, as a control, the chromosomal DNA of normal cells was pre-digested with DNase I and then assessed by TUNEL reaction mixture to show the nuclear morphology. Figure 5 showed nuclei observed in normal fungal cells. Fungal cells treated with 2 mM aspirin showed a few TUNEL positive cells and condensed nuclei were observed when the cells under the same condition were stained byEffect of fungal culture age on ganoderic acids inductionTo produce a maximum amount of GAs, fungal mycelium from different culture stages was tested for GA production after induction with aspirin. Fungal mycelium was cultured on PDA for 4 to 12 days and then was incubated with 4 mM aspirin for 1 day. As shown in Figure 3B and 3C, GA production by the control culture increased with the cultivation time. Furthermore, GA 24 production and total GA production were both significantly enhanced by aspirin across the various ages of fungal mycelium. Maximal GA24 production and total GA production were 515.2 and 5385 mg/100 mg dry weight (DW), respectively, and this was obtained using the 12-days old mycelium. This is a 2.7-fold and 2.8-fold increase compared with the control. In comparison,Enhanced GA Production by Apoptosis in G. lucidumFigure 2. Time course of ganoderic acids and fungal biomass production of Ganoderma lucidum incubated with aspirin. Fungal mycelium was cultured on PDA for 4 days and then incubated with 4 mM aspirin for additional 6 to 48 hr. Fungal biomass (A), accumulation of lanosta-7,9(11), 24-trien-3a-o1-26-oic acid (ganoderic acid 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control group. doi:10.1371/journal.pone.0053616.gFigure 3. Effect of fungal culture age on ganoderic acids induction by aspirin in Ganoderma lucidum. Fungal mycelium cultured on PDA for 4?2 days was incubated with 4 mM aspirin for additional 1 day. Fungal biomass (A), lanosta-7,9(11), 24-trien-3a-o1-26oic acid (ganoderic acid 24) production (B) and total ganoderic acids (total GAs) production (C) by Ganoderma lucidum were determined. The means of three independent samples with standard deviations are presented. *p,0.05, **p,0.01, ***p,0.001 as compared with the control g.
