Gsk3787

The 4T1 cells (ATCC) were incubated with RPMI-1640 medium (Gibco, Carlsbad, CA) containing 10% fetal bovine serum in an incubator at 37 °C and 5% CO₂. The PPAR-δ in 4T1 cells was antagonized using different concentrations (1, 5, 10, 20, 40 or 80 µM) of the PPAR-δ antagonist GSK3787 (S8025, Selleck). Total protein extracts were obtained using a total protein extraction kit (KeyGEN Biotech, Jiangsu, China) according to the manufacturer's instructions. Protein concentrations were measured using a BCA protein assay (23227, Thermo Fisher Scientific) according to the manufacturers' protocols to ensure equal protein loading for each sample.

Human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany) and grown in endothelial cell growth medium (PromoCell) supplemented with gentamycin (50 µg mL⁻¹) and amphotericin B (50 ng mL⁻¹). For all experiments, we used HUVECs pooled from up to four donors, which did not exceed passage 4. Human embryonic kidney (HEK) 293 cells (ATCC CRL-1573) were grown in DMEM medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS), 100 IU mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin (Invitrogen, Cergy Pontoise, France). C166 mouse endothelial cells (accession number CRL-2581) and LLC1 mouse lung cancer cells (accession number CRL-1642) were grown in DMEM medium (Invitrogen, Cergy Pontoise, France). Media were supplemented with 10% fetal calf serum (FCS), 100 IU mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin. As positive control for apoptosis assays, LLC1 mouse lung cancer cells were treated with 100 nmol/L Staurosporine (Sigma, St. Louis, MO, USA) overnight. For RNA isolation and quantitative RT-PCR experiments, HUVEC and LLC1 cells were maintained for 48 h (HUVEC) or 24 h (LLC1) in medium in the presence of GW0742 (Selleckchem, Houston, TX, USA) or GSK3787 (Selleckchem) dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 µmol/L. Controls were treated with vehicle (0.1% DMSO) only [6 (link),16 (link)].

Recombinant rat IL-1β (Peprotech), PPARδ (abcam, ab23673), Aggrecan (abcam, ab3773), Collagen II (abcam, ab239007), MMP13 (abcam, ab39012), SOX9 (abcam, ab185966), Cleaved-PARP (Cell Signaling Technology, Asp214), Cleaved-caspase3 (Cell Signaling Technology, Asp175), Bcl-2 (abcam, ab194583), Bax (Cell Signaling Technology D2E11), ATG5 (abcam, ab108327), Beclin1 (abcam, ab62557), LC3 (Sigma-Aldrich, L8918), GW501516 (Selleck, S5616), GSK3787 (Selleck, S8025).