Extended Data Fig. 5: Validation of knockout and knockdown cell lines.
(a) Analysis of whole cell lysates (WCL) by SDS-PAGE and western blotting for NIX/BNIP3 double-knockout clones #6 and #10, with and without induction of mitophagy by 24 h of DFP treatment. Results are representative of two biologically independent replicates. (b) Mitophagy flux measured by flow cytometry of wild-type (WT) or NIX/BNIP3 double-knockout (DKO) HeLa cells (clone #6), left untreated or treated with DFP for 24 h. Data are presented as mean ± s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. ns, not significant. (c) Analysis of knockdown efficiency for ATG13. HeLa cells were transfected 72 h prior to the FACS experiment, with the addition of DFP for the last 24 h to induce mitophagy, and analysed by flow cytometry. Cells were collected after the experiment and analysed with SDS-PAGE and western blotting. We selected the concentration of 10 nM for the FACS experiments throughout this manuscript. Results are representative of two biologically independent replicates. (d) As in (c), but for HeLa cells transfected with siRNAs against FIP200, ULK1 or scrambled as a control (−). Results are representative of two biologically independent replicates. Source numerical data, including exact P values, and unprocessed blots are available in source data.
