Figure 2.

Zafirlukast induces metabolically active brown adipocytes. (a) Quantification of the dose–response effect of zafirlukast in brown adipocyte differentiation based on HCI data (b) High-content imaging of differentiated SGBS cells treated with 2.2 μM zafirlukast. Fixed cells were stained with LipidTox (green) for neutral lipid droplets, anti-UCP1 antibodies (red), and Hoechst (blue) to visualize nuclei. (c) Gene expression of markers for brown adipocytes (UCP1) and adipogenesis (FABP4) in zafirlukast-treated cells, measured by qPCR. (d) Western blot analysis and quantification of UCP1 and FABP4 in SBGS cells treated with rosiglitazone, Ki16198 (LPA antagonist), and zafirlukast. LNCaP prostate cancer cell line was used as a positive control for UCP1 since it is known to express this protein. (e) Cellular respiration was analyzed using an XF96 extracellular flux analyzer as described in Methods. Oxygen consumption rate of zafirlukast and rosiglitazone over time was measured by the interference of mitochondrial pathways with specific inhibitors in the absence or presence of forskolin as a surrogate measure of cAMP-induced uncoupled respiration. (f) Quantification of uncoupled respiration. (c,f) *** p < 0.001; ** p < 0.01; * p < 0.05, one-way ANOVA.