Fig. 2: Mitochondrial Translocation of FPC ICD₁₅.

a Schematic diagram of mouse and human ICD₁₅ constructs used in the study, with the MTS sequences and the predicted p(Import) to mitochondria shown. The mutant amino acids are shown in red. b–d FPC ICD₁₅ constructs tagged with GFP were transfected into mIMCD3 cells and imaged. Mitochondria are shown in red, and FPC-GFP constructs in green. A field of cells is shown in the first panel, scale bar 50 µm. Box highlights area of magnified cells. Scale bar 20 µm. Three separate experiments per construct were performed independently with similar results. e Schematic of the split-GFP β-barrel complementation (upper panel). FPC ICD₁₅ fragment reconstitutes GFP barrel if mitochondria localization is achieved (lower panel). Figure created in BioRender. f–j Images of GFP₁₁-fused FPC ICD₁₅ fragments and GFP_1-10-fused mitochondria-localized mCherry in mIMCD3 cells. Leftmost image shows wide view merge, scale bar 10 µm. The three rightmost panels (GFP, mCherry, Merge) show a magnified image of the box in the first panel, scale bar 5 µm. Mouse and human ICD₁₅ split GFP constructs used are indicated on the left. Panel j shows a GFP₁₁-only Control. Three separate experiments per construct were performed independently with similar results. k Western blot of MDCK cell fractions from cells expressing mouse FPC C-terminal construct TMCT, spanning amino acids 3852–4059, with HA and V5 tags at the N- and C-termini, respectively. T total protein, C cytoplasmic fraction, M mitochondrial fraction. FPC was detected using E1 antibody. MDCK cells containing pcDNA5 vector were used as a negative control. Tubulin and TOM20 are loading controls. ICD₁₅ (“d”), ICD₁₂ (“e”), and ICD₆ (“f”) are indicated. kDa sizes correspond to less accurate commercial protein ladder. Three independent experiments per construct were performed with similar results. Source data are provided as a Source Data file.