Sulforaphane sfn

Validated U–373 MG, U–87 MG, and MDA–MB–231 cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 4 mM L–glutamine (Gibco ID 25030081, Waltham, MA, USA), 80 mg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain), and 1% amphotericin B solution (Lonza ID 17–836E, Basel, Switzerland), in 5% CO₂ at 37 °C. Sulforaphane (SFN) was purchased from LKT Laboratories (ID S8044, St Paul, MN, USA). Paraquat (PQ) was purchased from Sigma Aldrich (ID M2254, St. Louis, MO, USA). For synchronization at G1/G0, cells were FBS–deprived for 64 h and then replated in a standard medium (0 h point). For synchronization before S entry, a double thymidine pulse was performed. Cells were treated with 2.5 mM thymidine (Abcam, ID ab143719, Cambridge, UK), and then washed with PBS twice and maintained in thymidine–free medium for 8 h. Then, a second thymidine pulse was performed under the same conditions, and cells were finally chased to standard medium (0 h point). For synchronization in the M phase, cells were treated with 75 ng/mL nocodazole (Sigma Aldrich, ID M1404) for 16 h. Then, round and partially detached cells were harvested and plated in a nocodazole–free medium (0 h point).

Dimethyl sulfoxide (DMSO) and sulforaphane (SFN) were purchased from LKT Laboratories (St. Paul, MN, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). The anti-COX2, anti-iNOS, anti-p38, anti-ERK, anti-JNK, anti-actin, anti-HO1, anti-NQO1 and anti-Akt primary antibodies and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho-JNK, anti-phospho-ERK, anti-phospho-p38, anti-Nrf2 and anti-phospho-Akt primary antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sulforaphane (SFN) (catalog no.: S8044, LKT Laboratories, Inc., St. Paul, MN) and Dimethyl Fumarate (DMF) (catalog no.: 242926, Sigma-Aldrich, St. Louis, MO) and epigallocatechin gallate (EGCG) (catalog no.: E4143, Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO). Azidothymidine (AZT) (AIDS Reagent Program, Division of AIDS, NIAID, NIH Germantown, MD) and epigallocatechin gallate (EGCG) (catalog no.: G6817, LKT Laboratories, Inc., St. Paul, MN) were dissolved in water (50 mM stock).

Iohexol (low-osmolarity nonionic CM, 350 mg iodine/mL, GE Healthcare, Shanghai, China), SB (purity >98.0%, kindly donated from Shanghai Green Valley Pharm. Co., Ltd.), wortmannin (Abcam Inc., Cambridge, MA, USA), sulforaphane (SFN, LKT Laboratories Inc., St. Paul, USA), Nrf2 siRNA (Santa Cruz, CA, USA), control siRNA (Santa Cruz, CA, USA), Opti-MEM I (Invitrogen, CA, USA), and Lipofectamine 2000 solution (Invitrogen, CA, USA) were used in this study.