MCU is involved in nucleoid morphology and distribution. (A) Cells were treated with siRNAs for 96 h and then observed using an LSM710 confocal microscope. Representative images are shown. The white dashed square indicates the magnified area. (B) Protein levels in the MCU-knockdown cells were determined by immunoblotting using the indicated antibodies. (C, D) The maximum nucleoid size (C) and nucleoid number (D) in each cell of (A) were quantified using ImageJ. (E) The relative mtDNA copy number in MCU-knockdown cells was measured using quantitative PCR. (F) Cells were treated with indicated siRNA for 96 h and then fixed. After that nucleoids were stained with an anti-DNA antibody, indicating nucleoid, and observed under an LMS710 confocal microscope. The representative images are shown. The overlay images of nucleoids with mitochondria are shown in Supplementary Fig. S1A. (G–I) Cells were treated with the indicated siRNA for 96 h and then transfected with MCU-FLAG. Subsequently, cells were fixed and co-stained with an anti-DNA antibody, an anti-cytochrome c antibody, and an anti-FLAG antibody, indicating the nucleoid, mitochondria, and MCU-FLAG, respectively. Representative images obtained from an LMS710 confocal microscope are shown (G). The nucleoid images and overview images are shown in Supplementary Fig. S1B. The maximum nucleoid size (H) and nucleoid number (I) in each cell were quantified as in (C, D). All graphs show plots of the raw data and the mean ± SD. n ≥ 3 for each quantitative dataset. All statistical analyses were performed using Tukey–Kramer test. ^*P < 0.05, ^**P < 0.01.