β sitosterol

Immortalized human embryonic kidney cells (HEK293.T), human microglia (CHME3; a kind gift from prof. dr. M. Tardieu, Universite Paris-Sud, France^{66 (link)}), human oligodendrocytes (MO3.13), mouse neuroblastoma expressing APPswe (N2a/APPswe; a kind gift from prof. dr. T.W. Kim, Colombia University, USA^{67 (link)}), and monkey kidney cells (COS7) were used for in vitro experiments. All cell lines were cultured in DMEM (Sigma-Aldrich) containing 10% heat-inactivated FCS (Invitrogen, Merelbeke, Belgium) and 100 U penicillin/100 µg streptomycin/ml (Invitrogen), at 37 °C/5% CO₂. For phytosterol treatment, cells were incubated for 18 hours in culture medium without FCS containing the Eastern plants extracts, brassicasterol (Sigma-Aldrich), β-sitosterol, (Sigma-Aldrich), fucosterol (Sigma-Aldrich), stigmasterol (analytic confirmed purity of 99,9%), phytosterol mix (containing 60% β-sitosterol, 25% campesterol, and 15% stigmasterol; kindly provided by Ingmar Wester Raisio, Finland), T0901317 (Cayman Chemicals, Huissen, the Netherlands), ethanol (VWR), or DMSO (Sigma-Aldrich).

The MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan). The media was supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Shanghai, China), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific, Shanghai, China).
The cells were treated with 100 µg/mL F3, which was sourced from the Universiti Sains Malaysia Centre for Drug Research. The F3 was prepared from pulverized freeze-dried leaves of S. crispus as previously reported [29 (link),30 (link)]. MDA-MB-231 cells were also treated with 20 µM lutein, 25 µM β-sitosterol, or 90 µM stigmasterol (Cayman, MI, USA), based on the IC₅₀ concentrations obtained from the MTT assay [32 (link)]. Apigenin was used as a positive control for the detection of GLUT1 because it has been shown to inhibit both GLUT1 mRNA and protein expression [106 (link),107 (link)]. Meanwhile, tamoxifen was used as a positive control in all subsequent experiments because it has previously been reported to decrease PKC activity and affect the PI3K/AKT pathway in MDA-MB-231 cells [108 (link),109 (link)].

MNU was purchased from Sigma-Aldrich Co. St. Louise Mo 63103 USA. β-sitosterol was purchased from Cayman Chemical Company, Michigan, USA. All other chemicals were of analytical grade and obtained from Himedia Laboratories, Mumbai, India, else otherwise stated in the text.