Fig. 4.

p62zz is necessary for efficient EBSS-induced macroautophagy. a Schematic of LC3 tandem and Fis-tandem flux assay. b An example of the LC3 tandem flow cytometry assay under basal conditions or starved (5% serum media in 95% EBSS media) conditions in p62 WT MEFs, where a decrease in GFP expression can be observed and quantified to measure the percent of cells undergoing autophagy. c Number of cells undergoing autophagy 24 h after starvation in 5% serum media in EBSS: measured by the decreased ratio of mCherry/GFP via flow cytometry in p62-/- MEFS with stable expression of an empty vector, WT p62, D129K p62 or D147K p62 and stable expression of mCherry-GFP-LC3 tandem (representative figure of two experiments). d Quantification of (c), where the percent of cells undergoing autophagy was normalized to the p62-/- condition. One-way ANOVA was performed on two independent experiments. e Normalized percent of cells undergoing mitophagy measured by the decreased ratio of mCherry/GFP via flow cytometry in p62-/- MEFS with stable expression of an empty vector, WT p62, D129K p62, or D147K p62 and stable expression of mCherry-GFP-Fis1 tandem. Error bar in d and e represents s.e.m with N of 2. f Western blot analysis of mouse p62^fl/fl embryonic fibroblasts infected with GFP or CRE adenoviruses were reconstituted with retroviruses expressing WT Flag-p62 or point mutants of Flag-p62 treated with/without MG132 (0.2 µM, 24 h) in the presence or absence of hydroxychloroquine (10 µM, 24 h). Ratio between LC3-II and LC3-I quantified by densitometry using ImageJ. g Similar to f but with mouse embryonic fibroblasts expressing recombinant wild-type p62-flag or indicated point mutants