Cell Mediated Autophagy and Survival Supplementary Methods Renal Cell Lines Slr20 and Slr24 Were a Gift From

Staining Following incubation, 50 μl of 6% PFA (15714 EMS, Hatfield Pa) was added to a final concentration of 3% to each well for 12 minutes. Wells were washed 3x with PBS (50 μl volume per well of 384-well plate, inverting to remove washes). Plates were blocked and permeabilized in one step using 2% serum and 0.1% Triton X-100 in PBS at room temperature for 1 hour. Antibodies were added in PBS +2% serum. Primary antibodies were added overnight in 4C, then plates were rinsed 4x with PBS and secondary was added for 1hr covered, room temperature, on a slow orbital shaker. Hoechst 33342 (Molecular Probes/Invitrogen, California) was added at 1:10,000 with the secondary. Secondary antibodies utilized were Goat anti-Mouse Alexa 488/ 546, Goat anti-Rabbit 647, and Goat anti-Human 647. Actin was marked with Phalloidin (all from Molecular Probes, Invitrogen, California).