Extended Data Figure 3. Characterization of DELE1 submitochondrial localization.

(a-b) Zoom-out views for two-color 3D-STORM super-resolution images of DELE1-mClover vs. TOM20 and Hsp60. Two-color DELE1-mClover (magenta) vs. (a) TOM20 (green) or (b) Hsp60 (green), followed by the two separated color channels. Scale bars: 1 μm. The boxed regions correspond to Fig. 3g, h. Similar results were obtained in n = 3 independent experiments.

(c) Biochemical fractionation indicates that DELE1 associates with mitochondrial membranes.

Cells stably expressing DELE1-mClover were fractionated into cytosol and mitochondria. Mitochondria were incubated in either isotonic buffer (10 mM Tris HCl, pH 6.7, 0.15 mM MgCl₂ 0.25mM sucrose,1 mM DTT, protease inhibitor cocktail (Sigma #5892970001)) or H₂O (extreme hypotonic condition) for 5 min, followed by centrifugation (10000 xg for 10 min) to separate the supernatant and pellet. The pellet was either dissolved with RIPA buffer or incubated with 0.1M NaCO₃ (pH=11.4) for 30 min at 4°C. Supernatant and pellet from NaCO₃-treated samples were collected for WB. Unlike soluble matrix protein LonP1 and HSPD1, which can be extracted with H₂O incubation, only small proportion of DELE1 is present in the supernatant. NaCO₃ can extract the majority of the DELE1_S but not DELE1_L, which is similar to the pattern of mitochondrial membrane protein VDAC, suggesting that DELE1_L is more likely a membrane associated protein. n = 2 independent experiments.

For gel source data, see Supplementary Figure 1.

(d) The indicated DELE1-mClover constructs were transiently overexpressed in reporter cells, and reporter induction was quantified by flow cytometry (mean ± s.d., n = 3 culture wells). Subcellular localization was evaluated by microscopy in cells also expressing mitochondrial-targeted mRuby.

(e) Lack of co-localization of transiently expressed DELE1ΔN100-mClover and DELE1ΔN148-mClover (green) with the mitochondrial stain Mitotracker (red). Scale bar, 7 μm. Similar results obtained in n = 2 culture wells.

(f) Increased detection of DELE1-mClover outside the mitochondria upon oligomycin treatment. 3D-STORM super-resolution images of stably expressed DELE1-mClover (colors indicating depth in the z dimension) in untreated cells (left) and cells treated with 1.25 ng/mL oligomycin for 16 h (right). Areas boxed in red in the top panels are shown in higher magnification in the bottom panels. Similar results were obtained in n = 3 independent experiments.

(g) Co-localization of transiently expressed HRI-mClover and DELE1-mClover with the mitochondrial-targeted mRuby (Mito7-mRuby). Scale bar, 7 μm. n = 1 culture well.