mCherry-Parkin (mCh-Parkin) expressing WT, N/O (NDP52/OPTN) DKO and pentaKOs were quantified for a, GFP-LC3A, LC3B and LC3C translocation to mitochondria, b, GFP-WIPI1 or c, GFP-DFCP1 structures per cell (>100 cells counted for each sample) or d, were immunoblotted using phospho-specific anti-S757 and S317 ULK1 antibodies. e, mCherry-Parkin WT, N/O DKO and pentaKOs stably expressing GFP-ULK1 were quantified for GFP-ULK1 puncta per cell (left graph) and the percentage of those puncta on mitochondria (right graph). f, Representative data of e, cells were immunostained for Tom20 and GFP. g, pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCherry-ULK1 (mCh-ULK1) and myc-tagged receptors, were treated with rapalog then imaged live. h, Quantification of mitochondrial ULK1 puncta in g. i, Quantification of mitochondrial ULK1 puncta in pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCh-ULK1 and myc-OPTN mutants, treated with rapalog then imaged live. Quantification in a, b, c, e, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA. (***P<0.001, **P<0.005, *P<0.05, ns, not significant). For live cell quantification >75 cells counted in a blinded manner. Quantification in h and i were performed after removal of outliers, see Online Methods for details. OA, Oligomycin and Antimycin A. Scale bars, 10 μm. a.u., arbitrary units. See , and for representative images of a, b, c, and i respectively. See untreated samples of f and for untreated images of h and i.