Figure 1: Rubicon KO Starvation and Mitophagy HaloTag processing assay

Figure 1. Rubicon depletion promotes bulk autophagy and Parkin mitophagy. (A) LC3- HaloTag processing assay used to assess starvation autophagy in WT and Rubicon KO HeLa cells. Cells incubated in either complete growth medium or EBSS for 3 hours prior to collection and analysis. (B) Quantification of starvation autophagy flux in panel A. Autophagy flux was quantified as the fraction of HaloTag signal in the lower, processed band relative to total signal (n = 2 for rich conditions, n = 4 for starvation conditions). Significance scores derived from a one-tailed T-test. Error bars indicate standard deviation. (C) Su9-GFP-HaloTag processing assay used to assess mitophagy flux in Parkin expressing WT and Rubicon KO HeLa cells. Cells were either incubated in rich medium, or rich medium supplemented with 10 μM Oligomycin A and 5 μM Antimycin A for 6 H. (D) Quantification of mitophagy flux in panel C. Mitophagy flux was quantified as the fraction of HaloTag signal in the lower, processed band relative to total signal (n = 6 for WT and Rubicon KO). Significance scores derived from a one-tailed T-test. Error bars indicate standard deviation. All statistical replicates were drawn from independent experiments, and each lane contains lysates from independent experiments.