6 ohda

Neuroprotection assays were performed using alamarBlue^® reagent (Invitrogen, MA, USA). Briefly, SH-SY5Y cells were pretreated with SB for 1 h before adding 20 μM 6-hydroxydopamine (6-OHDA; Sigma, St. Louis, MO, USA) for 18 h. Cells were treated with LY294002 inhibitor (Sigma, St. Louis, MO, USA) or ZnPP (R&D Systems Inc., Minneapolis, MN, USA) prior to SB treatment for 1 h. Next, 10 μL alamarBlue^® was added after exposure to 6-OHDA, and optical density (OD) was measured with an ELISA reader. Relative protection (percent) was calculated as 100 × ((OD of the 6-OHDA + SB-treated cells − OD of the 6-OHDA-treated cells)/(OD of the control cells − OD of the 6-OHDA-treated cells)) as described previously [72 (link)].

6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). CellTiter-Glo^® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) and low-glucose DMEM were purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript^™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan). 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Merck KGaA (Darmstadt, Germany), and MitoSOX™ was obtained from Thermo Fisher Scientific (Waltham, MA). Antibody against actin (Code: SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-JNK (p46 and p54) (Code: #4668), phospho-p38 (Code: #4511), phospho-ERK (Code: #4376), and IκBα (Code: #4814) and goat anti-rabbit IgG, HRP-linked antibody (Code: #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare (Tokyo, Japan).

Ergothioneine was kindly provided by Tetrahedron (Paris, France). 6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Ergo-koji was provided by Euglena, Co., Ltd. (Tokyo, Japan). The ergothioneine content in Ergo-koji is 73.6 mg/100 g. Ergo-koji is fermented with euglena powder (Euglena Co., Ltd., Tokyo, Japan) in the fermentation stage of Rice-koji (Sakichi Co., Ltd., Nagasaki, Japan). The addition of euglena powder ensures a higher ergothioneine content of Ergo-koji than fermentation without euglena powder. MPP+ was purchased from Cayman Chemical (Ann Arbor, MI, USA). CellTiter-Glo^® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) was purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Merck KGaA (Darmstadt, Germany). The FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript^™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan).