On Western Chemiluminescent HRP Substrate (Millipore, MedChemExpress CP21 Billerica, MA, USA). All experiments were performed at least three times.Statistical analysisThe statistical analysis of GA production and biomass production across the various different treatments was carried out 
by Student’s t-test (Microsoft Excel, Seattle, WA, USA). Statistical significance was expressed as *p,0.05, **p,0.01, *** p,0.001.Results and Discussion Effect of aspirin on the production of ganoderic acids and fungal biomassTo evaluate the effect of aspirin on ganoderic acid production, BCRC 36111 was cultured on PDA for 4 days and then incubated with 0.5? mM aspirin for 1 day. Biomass production was gradually reduced as the aspirin concentration increased (Figure 1A). Incubation with 0.5 mM aspirin slightly increased total GAs production and lanosta-7,9(11), 24-trien-3a-o1-26-oic acid (ganoderic acid 24, GA 24) production. Higher doses of aspirin (1? mM) significantly increased GA production (Figure 1B and 1C). The time course of GA induction by 4 mM aspirin was studied further. A reduction in fungal biomass was observed after 6 hr treatment with 4 mM aspirin (Figure 2A). Incubation of the fungal mycelium with 4 mM aspirin for 6 hr slightly enhanced total GA production, but had no effect on GA 24 production. Administration of aspirin for longer than 12 hr resulted in enhanced GA 24 production and total GAs production (Fig. 2B and 2C).Figure 1. Effect of aspirin concentration on the accumulation of ganoderic acids and fungal biomass. Fungal mycelium was cultured on PDA for 4 days and then incubated with 0.5? mM aspirin for additional 1 day. Fungal biomass (A), accumulation of lanosta7,9(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.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). CB 5083 normal 
fungal cell did not show any fluorescent signal after TUNEL staining, indicating that 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 16574785 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.On Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). All experiments were performed at least three times.Statistical analysisThe statistical analysis of GA production and biomass production across the various different treatments was carried out by Student’s t-test (Microsoft Excel, Seattle, WA, USA). Statistical significance was expressed as *p,0.05, **p,0.01, *** p,0.001.Results and Discussion Effect of aspirin on the production of ganoderic acids and fungal biomassTo evaluate the effect of aspirin on ganoderic acid production, BCRC 36111 was cultured on PDA for 4 days and then incubated with 0.5? mM aspirin for 1 day. Biomass production was gradually reduced as the aspirin concentration increased (Figure 1A). Incubation with 0.5 mM aspirin slightly increased total GAs production and lanosta-7,9(11), 24-trien-3a-o1-26-oic acid (ganoderic acid 24, GA 24) production. Higher doses of aspirin (1? mM) significantly increased GA production (Figure 1B and 1C). The time course of GA induction by 4 mM aspirin was studied further. A reduction in fungal biomass was observed after 6 hr treatment with 4 mM aspirin (Figure 2A). Incubation of the fungal mycelium with 4 mM aspirin for 6 hr slightly enhanced total GA production, but had no effect on GA 24 production. Administration of aspirin for longer than 12 hr resulted in enhanced GA 24 production and total GAs production (Fig. 2B and 2C).Figure 1. Effect of aspirin concentration on the accumulation of ganoderic acids and fungal biomass. Fungal mycelium was cultured on PDA for 4 days and then incubated with 0.5? mM aspirin for additional 1 day. Fungal biomass (A), accumulation of lanosta7,9(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.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 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 16574785 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.
