Figure 3: HACE1 is required for autophagic clearance of Ub⁺ protein aggregates independent of its E3 ligase activity.

(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub⁺ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm. (b) Representative confocal micrographs of Hace1^−/− and Hace1^+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1^−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm. (c,d) WT and Hace1^−/− MEF as well as Hace1^−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P<0.001 (one-way analysis of variance). Scale bars, 10 μm.