Atorvastatin

OE33 cells were treated 24 h before leptin stimulation with expression plasmids containing DN-Ras, DN-cdc42, DN-RhoA and DN-Rac1 (cDNA Resource Centre, Bloomsburg University, Pennsylvania, USA), using previously described methodology [19 (link)]. Where appropriate, the prenylation inhibitors FTI276 and GGTI298 (both 10 μM) (both Tocris, Abingdon, Oxford, UK) were added 24 h prior to leptin stimulation [29 (link)]. In selected experiments, atorvastatin (0.1 μM) (Tocris) and mevalonate (100 μm) (Sigma) were initially added 72 h prior to stimulation with leptin, with a medium change with fresh reagents added after 48 h.

Cells were cultured in U-shaped, nonadherent 96-well plates (150 μL, 5.0 × 10³ per well) in StemSpan-ACF medium (#09855, Stem Cell Technologies) supplemented with 20% BIT9500 serum replacement (#9500, Stem Cell Technologies), CC100 (#02690, Stem Cell Technologies) mix of cytokines, 50 ng/mL VEGF (#AF-100-20, PeproTech), and penicillin with streptomycin (P4333-100ML, Sigma Aldrich). Compositions of other tested media are shown in Supplementary Table 1. After 48 hours, 50 μL of fresh medium was added; then, 50 μL of medium was changed every other day. On day 6 of culture, cells were stimulated with 7.5, 15, or 30 μmol/L atorvastatin (#3776, Tocris), 7.5, 15, or 30 μmol/L resveratrol (R5010-100MG, Sigma Aldrich), 125, 250, or 500 μmol/L acetylsalicylic acid (A5376-100G, Sigma Aldrich), 2.5, 5.0, or 10 μmol/L sulforaphane (S441-5MG, Sigma Aldrich), or 1.25, 2.5, or 5.0 mmol/L metformin (#317240-5MG, Sigma Aldrich). Conditioned media were collected after 48 hours. The controls, nonconditioned media, contained the same concentrations of stimulants and were kept for 48 hours in a cell culture incubator in the same conditions as stimulated cells. Wash-out conditioned media were collected from stimulated cells after washing with PBS and reseeding on 96-well plates in the complete fresh medium. DMSO, which was used to dissolve all stimulants, was added to the control wells.