Fig. 4

miPEP133 protein function. a The miPEP133-interacting proteins in HEK293 cells identified by mass spectrometry. b Representative confocal microscopy images of flag-tag-labeled miPEP133 in the cytoplasm of HEK293 and C666–1 cells. c Western blot images of miPEP133 in cellular fractions of HEK293 cells and co-IP of miPEP133 and HSPA9 in the mitochondrial fraction. TOM20, DNA topoisomerase 1 (TOP1), and GAPDH were used as loading controls for mitochondrial, nuclear, and cytoplasmic fractions, respectively. d Co-IP of HSPA9 and its interacting proteins demonstrated the ability of miEPE133 to block the interaction of HSPA9 to other proteins, including HSP60, TIM44, and VDAC1. e Representative images of western blot of mitochondrial proteins in from the control HEK293 cells and the miPEP133-overexpressing HEK293 cells. f Summarized flow cytometry result of JC-1-staining in HEK293 cells transfected with control or miPEP133 plasmid. CCCP-treated HEK293 cells were used as positive control. Student’s t-test, **p < 0.005. g Representative confocal microscopy images of HEK293 cells transfected with control vector or miPEP133-expressing vector. GFP (green) indicated the cells were transfected with the vectors. TOM20 (red) labeled the mitochondrial outer membrane. DAPI (blue) was used to stain the nuclei. Dotted circles indicate the cells with shrinking nucleus. h Cellular ATP level in control and miPEP133-expressing HEK293 cells. Two-way ANOVA, #p < 0.0001. i Schematic model of miPEP133-regulated mitochondrial integrity and function