Fig. 2

Mito-riboBiFC detects translation-dependent assembly of mitoribosomes. a Schematic design of mito-riboBiFC. Mitochondrial ribosomal proteins MRPL2 and MRPS6 were used as a BiFC pair for the mito-riboBiFC and illustrated with porcine 55S mitoribosome cryo-EM structure [28]. MRPS16 and MRPL50 served as a negative control. b Representative images of mito-riboBiFC (top) and mVenus-detectable GFP antibody staining (bottom) in Neuro2A cells. Mito-mTFP1 and MT-CO1 were used as mitochondrial markers (scale bar, 2 μm). c Quantification of the fluorescent mito-riboBiFC signals in panel b. Mito-riboBiFC signals were normalized to the average of fluorescent signals in S6-VN/L2-VC group. Data represent mean ± SEM (N = 3 replicates and n = 100–143 cells). ****P < 0.0001, as determined by aligned ranks transformation ANOVA with Wilcoxon rank-sum test. d Quantification of signal intensity of GFP antibody staining in b. GFP signals were normalized to the average of fluorescent signals in S6-VN group. Data represent mean ± SEM (N = 3 replicates and n = 41–76 cells). n.s., not significant; ****P < 0.0001, as determined by aligned ranks transformation ANOVA with Wilcoxon rank-sum test. e Pseudo-color images of mito-riboBiFC after sequential treatment of puromycin (Puro) and chloramphenicol (CA) (scale bar, 2 μm). f Quantification of the mito-riboBiFC signals in e. Line plot shows the intensity changes of mito-riboBiFC. A Vehicle-CA group was compared with other groups at each time point for the statistical test (left panel). Dot plot displays the relative intensity of the mito-riboBiFC 90 min after CA treatment (1st vehicle, water; 2nd vehicle, ethanol). Data represent mean ± SEM (N = 3 replicates and n = 22–26 cells). Aligned ranks transformation ANOVA detected significant interaction effects of Puro and CA on the mito-riboBiFC intensity at the 90-min time point (P < 0.0001). **P < 0.01, ****P < 0.0001, as determined by Wilcoxon signed-rank test (left panel) or Wilcoxon rank-sum test (right panel)