Fig. 4: Mitochondria are ubiquitinated during Rab32-LRO-mediated mitophagy.

A BMDMs were treated with OA for 6 h. Following treatment, cells were fixed, stained with anti-Tomm20 and anti-ubiquitin antibodies. White arrowheads indicate ubiquitin-positive mitochondria. Scale bar: 10 μm. Each data point represents the number of ubiquitin signals per image, normalized to the number of cells. N = 5 images for “-” and 15 images for “OA”. Images were analyzed using the ImageJ ‘Find Maxima’ tool, applying a defined threshold in a blinded manner. The experiments were performed twice, and a representative result is presented. Data are presented as mean values ± SD. B WT or DKO BMDMs were treated and analyzed as in (A). Scale bar: 5 μm. Each point represents the number of ubiquitin signals per image, normalized to the number of cells. N = 4 images for each group. The experiments were performed twice, and a representative result is presented. Data are presented as mean values ± SD. C Atg7 KO or Fip200 KO RAW264.7 cells expressing GFP-Rab32 were pre-stained with MitoTracker and treated with OA and bafilomycin A1, with or without TAK243 (5 μM) for 3 h. Each dot represents the fluorescence intensity of MitoTraker inside Rab32 rings (N = 199, 196, 230, 203 rings from left to right), measured using ImageJ. Similar experiments were conducted at least twice, and a representative result is shown. Box plots indicate the median (center line), the interquartile range (box), and the range (whiskers). D Atg7 KO RAW264.7 cells expressing GFP-Rab32WT or QL were treated with the same condition in Fig. 3F, with or without TAK243 for 1.5 h. White arrows indicate the MitoTracker positive signal engulfed into Rab32-LRO. E Atg7 KO RAW264.7 cells expressing GFP-Rab32 were stained with MitoTracker and then treated with OA and bafilomycin A1 with or without TAK243 for 3 h. After fixation, cells were stained with the anti-p62 antibody. Yellow arrows indicate p62-positive mitochondria inside Rab32-LRO. White arrowheads indicate p62-positive mitochondria associated with Rab32-LRO. Scale bar: 5 μm (upper), 2 μm (bottom). F Scramble control or p62 knockdown (KD) WT or Atg7 KO RAW264.7 cells were transduced with GFP-Rab32 using a retrovirus. After 24 h of transduction, cells were stained with MitoTracker and treated with OA and bafilomycin A1 for 3 h. Scale bar: 2 μm. The percentage of MitoTracker-positive Rab32-LROs per cell was quantified. N = 20, 20, 18, 19 cells from left to right. The experiments were performed twice, and a representative result is presented. Data are presented as mean values ± SD. Statistical significance was analyzed using an unpaired two-tailed t-test (***p < 0.001; ****p < 0.0001).