S4441

(1)

The measurement of glutathione (GSH) content: Briefly, 1 × 10⁶ cell lysates were determined using a commercial GSH and GSSG Assay Kit (S0053, Beyotime, China) according the manufacturer's instructions as described above [42 (link)].

(2)

The measurement of intracellular Fe²⁺ content: The experiments were performed as described previously [47 (link)]. Briefly, 1 × 10⁶ cells were washed with cold PBS for 3 times, collected, suspended and incubated with 1 μM ferrous iron probe FerroOrange (F374, Dojindo, Japan) for 15min followed by flow cytometric analysis (Ex: 561 nm/Em: 570–620 nm).

(3)

Lipid peroxidation analysis: The experiments were performed as described previously [42 (link)]. Briefly, 1 × 10⁶ cells were incubated with 5 μM BODIPY-C11 (D3861, Invitrogen, USA) for 30 min at 37 °C. Cells were washed 3 times with PBS, harvested and suspended in serum-free medium followed by flow cytometric analysis (Ex: 488 nm/Em: 510–555 nm).

For treatment of sorafenib (5 μM), sulforaphane (5 μM, S4441, Sigma, USA) or alkaloid trigonelline (0.5 μM, T5509, Sigma, USA), cells were treated by these agents for another 24 h before the measurement.

The human colorectal adenocarcinoma cancer cell lines Caco-2 and CX-1 were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. Caco-2 and CX-1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 (Gibco, Thermo Fisher Scientific), respectively, supplemented with 10% fetal bovine serum (BI, Israel) and 1% penicillin/streptomycin (PEN/STREP 100×, MILLIPORE, USA). Cells were cultured at 37°C in a 5% CO2 and 95% air humidified incubator. Stocks of SAL (S4526, Sigma-Aldrich, USA) and SFN (S4441, Sigma-Aldrich, USA) were prepared via dissolution in dimethyl sulfoxide (DMSO, D2650, Sigma-Aldrich, USA) at concentrations of 50 mM and 100 mM, respectively, and the solutions were stored at −20°C. The final concentration of DMSO was less than 0.1%. Antibodies against PI3K (1:1000 dilution), Akt (1:1000 dilution), phosphorylated Akt (Ser473) (1:1000 dilution), p53 (1:1000 dilution), cleaved poly ADP-ribose polymerase (PARP) (1:1000 dilution), and β-actin (1:1000 dilution) as well as the PI3K inhibitor LY294002 were purchased from Cell Signaling Technology. Antibodies against Bcl-2 (1:100 dilution) and Bax (1:100 dilution) were obtained from Santa Cruz Biotechnology.

3 - (4 (link), 5 (link)- d i m e t h y l t h i a z o l- 2 (link)- y l) - 2 , 5-diphenyltetrazolium
bromide (MTT) (Alfa Aesar by Thermo Fisher Scientific, England) assay was conducted to
evaluate the cytotoxicity of SFN (S4441, Sigma-Aldrich, USA) and to determine its optimal
dose for treating cells. First, SFN (stock concentration: 5 mg/mL) was diluted in DMSO
(Sigma-Aldrich, USA) and different concentrations of it were prepared by diluting in the
culture medium including 5, 10, 15, 20, 25, and 30 µM. Then, GCs were seeded on a 96-well
plate at a density of 1×10⁴ cells per well and exposed to the mentioned doses
of SFN for 24 hours. Furthermore, we also pretreated GCs with the mentioned concentrations
of SFN for 22 hours and then exposed them to 200 µM of H₂ O₂ for 2
hours to determine an optimal dose of SFN for protecting cells against H₂ O₂ induced OS. Next, 0.5 mg/ml of MTT solution was added to the wells and the
cells were incubated at 37°C for 4 hours in dark. Then the produced colorful crystals were
dissolved in DMSO and, the optical density (OD) was measured at 570 nm wavelength using a
microplate reader (BioTek, USA).