Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

Various peroxisome proliferator‐activated receptor (PPAR)‐γ agonists differently induce differentiation of cultured human keratinocytes

“…However, ciglitazone and troglitazone were used at a boundary concentration around 5-10 lM that can induce both on-and off-target effects (23). As a result, predominance of off-target effects over PPAR-c activation might occur, since ciglitazone and troglitazone have relatively low PPAR-c activation capability according to PPAR-c binding affinities (measured EC50 values); that is, rosiglitazone > pioglitazone > troglitazone > ciglitazone (24,25). The lack of loricrin upregulation after ciglitazone or troglitazone treatment may be due to the off-target effects followed by p21 expression, which suppress loricrin expression more strongly than filaggrin (20).…”

Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ

“…The PPAR␥ CALUX cell line was obtained from BioDetection Systems B.V. (Amsterdam, the Netherlands). PPAR␥ CALUX cells are based on human osteoblastic (U2-OS) cells (American Type Culture Collection [ATCC], Manassas, VA) stably transfected with a human PPAR␥ expression plasmid and a luciferase reporter construct (22). The cells were cultured as described previously in a 1:1 mixture of DMEM and Ham's F-12 medium (Invitrogen, Breda, the Netherlands) supplemented with 7.5% fetal bovine serum (FBS) (Invitrogen), 1% nonessential amino acids (Invitrogen), and penicillin-streptomycin (Invitrogen) to final concentrations of 10 U/ml and 10 g/ml, respectively (22,23).…”

“…The PPAR␥ CALUX assay was performed as described previously (22,24). In short, for the exposure experiments, PPAR␥-CALUX cells were plated in the 96-well plates with phenolred-free 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 5% dextran-coated charcoal-stripped FBS (Invitrogen) at a volume of 200 l per well.…”

Novel targets for the development of drugs for Type 2 Diabetes Mellitus

“…The cell line that was used for this study is a CALUX reporter gene cell line consisting of U2OS cells stably transfected with a 6xPPREtataLuc construct and an expression vector specific for PPARγ2 described in detail elsewhere (Gijsbers et al, 2011). Cells of this PPARγ2 CALUX reporter cell line were cultured in Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM with high glucose and l-glutamine)(Glutamax, Invitrogen, Breda, Netherlands) supplemented with 7.5% Fetal Calf Serum (FCS, Invitrogen), 1% NonEssential Amino acid (NEAA, Invitrogen) and 0.1% penicillin/streptomycin (10u/ml10μg/ml, Invitrogen).…”

“…On the second day cells were exposed to TZDs at concentrations of 100 pM up to 10 μM for 24h with the exception of DRF-2189 that was tested at concentrations of 1nM up to 10 μM. On the third day, cells were lysed and luciferase activity was measured as described before (Gijsbers et al, 2011). To this end 100 μl of Flash Mix (20 mM tricine, 1.07 mM (MgCO 3 ) 4 Mg(OH) 2 5H 2 O, 2.67 mM MgSO 4 , 0.1 mM EDTA, 2.0 mM dithiothreitol, 470 μM D-luciferine, and 5.0 mM ATP) were added per well and light production was measured directly using a luminometer (Anthos, Eugendorf, Austria) and expressed as relative light units (RLUs).…”