Was SIS3 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 GHRH (1-29) 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 group. doi:10.1371/journal.pone.0053616.geither TUNEL or DAPI. A large number of TUNEL positive cells were observed in fungal cells incubated with 3 mM aspirin, and a condensed nuclear morphology was also presented (Figure 5). However, a high background level of DAPI staining was present in the fungal cells treated with 3 mM aspirin (data not shown). To our knowledge, this is the first report showing that aspirin is able to induce apoptosis in G. lucidum. Our result suggests that GA biosynthesis occurs during cell 1081537 apoptosis in G. lucidum. Previous studies have shown that secondary metabolite biosynthesis in fungi is coordinated with fungal development and is regulated by environment factors, including nutrition, pH, light and temperature [26]. Environmental and developmental cues then mediate secondary metabolites biosynthesis via a range of transcription factors and various signal transduction pathways such as heterotrimeric G-protein signaling, cAMP signaling, Ras family GTPase signaling and MAPK signaling [27,28]. To the best of our knowledge, this study is the first to indicate that apoptosis signaling is correlated to fungalsecondary metabolite biosynthesis. Other medicinal fungi such as Inonotus obliquus, Poria cocos, Antrodia cinnamomea and other Ganoderma species, have also been used as folk remedies for many centuries, and triterpenoids has been proved to be the functional components in these fungi [1,29?1]. However, the regulation of triterpenoid biosynthesis in these fungi remains unknown. It is possible that apoptosis signaling regulates triterpenoid biosynthesis in these medicinal fungi too. In this context, apoptosis induction may have great practical value in the functional food industry where these fungi are used to produce functi.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 group. doi:10.1371/journal.pone.0053616.geither TUNEL or DAPI. A large number of TUNEL positive cells were observed in fungal cells incubated with 3 mM aspirin, and a condensed nuclear morphology was also presented (Figure 5). However, a high background level of DAPI staining was present in the fungal cells treated with 3 mM aspirin (data not shown). To our knowledge, this is the first report showing that aspirin is able to induce apoptosis in G. lucidum. Our result suggests that GA biosynthesis occurs during cell 1081537 apoptosis in G. lucidum. Previous studies have shown that secondary metabolite biosynthesis in fungi is coordinated with fungal development and is regulated by environment factors, including nutrition, pH, light and temperature [26]. Environmental and developmental cues then mediate secondary metabolites biosynthesis via a range of transcription factors and various signal transduction pathways such as heterotrimeric G-protein signaling, cAMP signaling, Ras family GTPase signaling and MAPK signaling [27,28]. To the best of our knowledge, this study is the first to indicate that apoptosis signaling is correlated to fungalsecondary metabolite biosynthesis. Other medicinal fungi such as Inonotus obliquus, Poria cocos, Antrodia cinnamomea and other Ganoderma species, have also been used as folk remedies for many centuries, and triterpenoids has been proved to be the functional components in these fungi [1,29?1]. However, the regulation of triterpenoid biosynthesis in these fungi remains unknown. It is possible that apoptosis signaling regulates triterpenoid biosynthesis in these medicinal fungi too. In this context, apoptosis induction may have great practical value in the functional food industry where these fungi are used to produce functi.
