Butylhydroxyanisole bha

Fresh Spanish mackerel (Scomberomorous niphonius) was purchased from Zhenhua Supermarket in Yantai, China. Natural whey protein (NWP, 95%) was bought from Davisco Foods International, Inc. in Minneapolis, MN, USA. 1,1,3,3-tetraethoxypropane, trichloroacetic acid (TCA), ethylene diamine tetra acetic acid (EDTA), and butylhydroxyanisole (BHA) were purchased from Sigma Corporation, St. Louis, MO, USA. Tris-HCl and Sodium dodecyl sulfate (SDS) were purchased from Beijing Dingguo Chansheng Bio-Technology Co., Ltd. in Beijing, China. Additionally, all the other chemicals (NaCl, HCl, ethyl acetate, ethanol) used in the experiment were of analytical grade and bought from Sinopharm Chemical Reagent Co., Ltd. in Shanghai, China.

MSCs were seeded at a density of 5 × 10⁴ cells/cm² and neural differentiation was performed with modifications to the procedure described previously (Woodbury et al., 2000) when reached approximately 50% confluence. Briefly, MSCs were incubated in the medium consisting of 10% FBS and 10 ng/ml basic fibroblastic growth factor (bFGF; Sigma) in L-DMEM for 24 h (referred to as 24-preinduction), transferred to neural induction medium containing 200 mM butylhydroxy anisole (BHA; Sigma) dissolved in 2% dimethylsulfoxide (DMSO; Sigma) for 5 h (5-h induction), and maintained with H-DMEM containing 1% N2 (Gibco) for 18 and 48 h (18- and 48-h maintenance) respectively.

Antibodies against the following proteins were used: ATG5 (#0262-100/ATG5-7C6) from Nanotools (Teningen, Baden-Württemberg, DE), ATG7 (#8558) ), Cleaved-Caspase3 (#9664), LAMP-1 (#9091) from Cell Signaling Technology (Danvers, MA, USA), PARP-1 (C-2-10) (#BML-SA249-0050) from Enzo Life Sciences (Farmingdale, NY, USA), Actin β (#NB600-501) from Novus Biologicals (Centennial, CO, USA), HRP conjugated rabbit (#111-035-003) and HRP conjugated mouse (#115-035-174) from Jackson ImmunoResearch (Ely, Cambridgeshire, UK), Alexa488 conjugated anti-rabbit (#A11008) from Invitrogen (Waltham, MA, USA). Hoechst 33258 (#14530), E64d (#E8640), Propidium iodide (#P4864), Chloroquine diphosphate salt (#C6628), QV-D-Oph (#SML0063), butylhydroxyanisole (BHA, #B1212000), N-acetyl-L-cysteine (NAC, #A8199) were purchased from Sigma-Aldrich (St. Louis, MO, USA).). Tetramethylrhodamine methyl-ester, TMRM (T-668) was purchased from Molecular Probes (Eugene, OR, USA). Fluoromount G (#00-4958-02) was purchased from Invitrogen. 20A was synthesized as previously described [42 (link),43 (link)]. Ferrostatin-1 (#341494) was purchased from Calbiochem (San Diego, CA, USA)), while LLOME (#16008) and Erastin (#17754) were purchased from Cayman Chemical (Ann Arbor, MI, USA).