Journal: bioRxiv
Article Title: The Cancer/Testis Antigen FATE1 Antagonizes Fission and Preserves Mitochondrial Network Integrity under Cytotoxic Stress
doi: 10.1101/2025.08.18.670752
Figure Lengend Snippet: a Sequence alignment of the C-terminal regions of human FATE1 and Mff generated with Jalview using the T-Coffee algorithm under default settings. Amino acid numbering corresponds to Mff. b Coiled coil domain prediction of FATE1 using the COILS program. Mff and Bnip3 were included as positive and negative controls, respectively. Predicted probabilities for coiled coil formation are shown for scanning windows of 14, 21 and 28 residues. c Schematic comparison of the domain architecture of Mff and FATE1, highlighting sequence homologies, including the predicted coiled coil domain and transmembrane region. R1 and R2 indicate the short repeat motifs in Mff involved in Drp1 recruitment. d Representative widefield images of GFP-Ctrl, GFP-FATE1, or GFP-Mff co-expressed with RFP-KDEL (ER marker) for 24 h in HeLa cells. Blue arrows, ER-localized FATE1 at perinuclear regions. e Representative widefield images of GFP-Ctrl, GFP-FATE1, or GFP-Mff co-expressed with mito-RFP (mitochondrial marker) for 24 h in HeLa cells. f Subcellular fractionation of HeLa cells transfected with GFP-tagged constructs as indicated. Fractionation yielded cytosolic (S1), rest-mitochondrial (P1) and purified mitochondrial (P2) fractions, analyzed by immunoblotting. g Representative widefield images of MCF-7 cells expressing GFP-FATE1 or GFP-Mff for 24 h and IF stained for mitochondrial matrix protein TRAP1. Plot profiles along yellow lines in zoom images show fluorescence intensity of GFP and TRAP1 signals. h STED super-resolution microscopy of HeLa cells expressing pcDNA-FATE1 and IF stained for FATE1 in combination with Cox IV (IMM marker) or Tom20 (OMM marker). Plot profiles show fluorescence intensity of FATE1 and Cox IV or Tom20. Scale bars, 10 µm
Article Snippet: Cells were then incubated with primary antibodies against Tom20 (Santa Cruz, no. sc-11415; 1:200) or FATE1 (Santa Cruz Biotechnology, no. sc-101220; 1:100) for 1 h at room temperature, or against COX IV (Cell Signaling, no. 4850S; 1:50) or TRAP1 (Novus Biologicals, no. NB300-555; 1:100) antibodies at 4°C overnight.
Techniques: Sequencing, Generated, Comparison, Marker, Fractionation, Transfection, Construct, Purification, Western Blot, Expressing, Staining, Fluorescence, Super-Resolution Microscopy
