Figure 1. MOTS-c Is a Mitochondrial-Encoded Peptide that Can Reside in the Nucleus.

(A) Immunoblots of endogenous MOTS-c from different subcellular fractions of resting HEK293 cells. W, whole-cell lysate; C, cytoplasm; N, nucleus; and M, mitochondria.

(B) Endogenous MOTS-c detection by immunofluorescence microscopy in resting HEK293 cells. The nucleus is outlined by white dashed lines.

(C) Immunoblot detection of endogenous MOTS-c in increasing levels of nuclear extracts from resting HEK293 cells. Antibody specificity to MOTS-c was confirmed by neutralizing peptide competition. Nuclear proteins such as lamin B1 and histone H2B were used as nuclear loading controls.

(D) Localization of exogenously treated FITC-MOTS-c peptide (1 μM; 30 min) in HEK293 cells by confocal microscopy.

(E and F) Subcellular distribution pattern of wild-type (WT) and mutant (₈YIFY₁₁ → ₈AAAA₁₁ and ₁₃RKLR₁₆ → ₁₃AAAA₁₆) MOTS-c tagged with EGFP in resting HEK293 cells by (E) confocal microscopy (EGFP-MOTS-c^WT, EGFP-MOTS-c^YIFY, and EGFP-MOTS-c^RKLR, respectively) and (F) immunoblotting of subcellular fractions (M^WT, M^YIFY, and M^RKLR, respectively). Representative images are shown (n = 3).

Cells were co-stained with DAPI (nucleus) and MitoTracker Red (mitochondria). Scale bar, 10 μm. See also Figure S1.