Effects of artificial tethering of the FKBP domain, interaction analysis of BNIP3/NIX with other autophagy factors, and serial ultrathin sections related to Fig. 7 G . (A and B) Representative immunofluorescence images of Atg13 and LC3 (A) and mt-Keima (B) in WT and B/N DKO cells, with or without artificial tethering of the FKBP domain to mitochondria. In merged images, mitolysosomes (excited at 590 nm) and mitochondria (excited at 430 nm) are indicated by red and green, respectively. Scale bars: 10 μm. (C) Quantification of the mitolysosomes in the cells shown in B. More than 200 cells were analyzed in each experiment. Data expressed as the means ± SEM of three independent experiments; ***P < 0.001 and not significant (NS), determined by one-way ANOVA followed by a Tukey–Kramer post hoc test. (D) Immunoprecipitation (IP) analysis of mCherry-BNIP3 or mCherry-NIX expressed in B/N DKO cells cultured with DFP for 12 h and then treated for an additional 12 h with 100 nM bafilomycin A1 and DFP. The cells were lysed, and IP was performed using RFP-trap magnetic agarose. Input is 10% of the IP fraction. (E) Serial ultrathin sections of the mitophagy profile. The framed image appears in Fig. 7 G. Section (s) numbers are indicated at the top right corner of each EM image. mt, mitochondrion; arrows, IM. Scale bars: 500 nm. Source data are available for this figure: SourceData FS4.