Figure 2.

Oleate (OLA), but not gonadotropin-releasing hormone (GnRH), induces mitochondrial superoxide production. LβT2 cells (RRID:CVCL_0398) cultured on Poly-l-Lysine (Sigma-Aldrich Inc.) coated Nunc Lab-Tek II chamber slides (Thermo Fisher Scientific) for 24 h were serum starved overnight, then exposed to either vehicle or 500 µM oleate for 2 h, then with vehicle or 10 nM GnRH for 30 min. Cells were subsequently treated with 5 µM red-fluorescent MitoSOX probe (Thermo Fisher Scientific) for 5 min. Afterward, cells were directly fixed with 2% paraformaldehyde solution for 15 min, washed, and cover slipped with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs) to visualize nuclei in blue. Blue DAPI and Red MitoSOX fluorescence was captured by wide-field fluorescent microscopy using Nikon TE2000-U microscope (Nikon America Inc., Melville, NY, USA) equipped with an X-Cite 120PC collimated light source (Lumen Dynamics Group Inc.) and a DAPI-1160A or mCherry-C000 filter set (Semrock, Inc.) using a CoolSNAP DYNO CCD camera (Photometrics Inc.). In LβT2 cells, GnRH alone does not enhance mitochondrial ROS production as determined by changes in red fluorescence, whereas OLA-treated cells showed a highly elevated signal. Co-treatment with GnRH and Oleic acid did not appear to alter overall staining intensity.