Figure 2
TNFR5 protein expression in INS-1 β-cells, high-fat-fed mouse islets, and human islets. (a) INS-1 cells incubated for 72 h in RPMI-1640 media supplemented with or without 28 mM glucose, 200 μM oleic acid, and 200 μM palmitic acid. Immunoblots were conducted using anti-TNFR5 primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and Li-Cor IR secondary antibody (Lincoln, NE, USA), and are representative of three independent experiments. (b) Imuunofluorescence of INS-1 cells fixed in 4% paraformaldehyde for 30 min, then permeabilized with 0.1% Triton X-100. Samples were treated with anti-DAPI (blue; Life Technologies Ltd., Paisley, UK) or anti-TNFR5 primary antibody (green), and incubated with secondary antibody conjugated to fluorescence dye (AF488; Life Technologies Ltd.). Images were taken with a Leica epi-fluo microscope (Wetzlar, Germany) using either a 20 × objective (upper panels) or 40 × objective (lower panels), and in each case are representative of three independent experiments. (c) C57Bl/6 mice were fed a high-fat or standard rodent diet for 10 weeks. Islets of mice were extracted and digested with collagenase, RNA extracted, reverse transcribed, and qRT-PCR analysis performed. Data were analysed using the ΔΔCT method from four independent experiments. (d) Human islets were cultured 24 h with or without 0.5 mM palmitate. RNA was extracted, reverse transcribed, and qRT-PCR analysis performed. Data were analysed using the ΔΔCT method from six independent experiments. *P<0.05
