Transmission Electron Microscopy of SKOV-3 Cells

For TEM, 6 × 10⁵ WT or NFE2L2/NRF2 KO SKOV-3 cells were seeded in six-well plates and, 24 hours later, processed as described previously (65 (link)). Briefly, cells were fixed in 2.5% glutaraldehyde + 2% PFA in 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) or Pipes buffer (pH 7.4) for 1 hour at room temperature. Samples were postfixed in 1% osmium tetroxide in Pipes buffer for 1 hour following a wash in 0.05 M Pipes buffer + 0.05 M glycine and 2 × 10 min washes in 0.1 M Pipes. Following two more 10-min washes in DIW, samples for TEM were block-stained in 2% aqueous uranyl acetate, washed in DIW, dehydrated through a graded series of alcohols, infiltrated with 1:1 alcohol and Spurr’s resin overnight, and then embedded in 100% Spurr’s resin overnight at 60°C. Sections were viewed using a FEI Tecnai Spirit electron microscope (FEI Company, Hillsboro, OR) operated at 100 kV. Eight-bit TIFF images were captured through an AMT 4-megapixel camera.