Fig 2: Upregulation of MICU1 in miR-181c/d-/- hearts. A, Quantitative PCR (SYBR) was performed from the RNA fractions of WT and miR-181c/d-/- hearts. Western blot analysis of MICU1 expression in heart tissue from WT (C57BL6/j) and miR-181c/d-/- mice. B, MICU1 (upper bands) expression was normalized to a-Tubulin (lower bands). Western blot analysis of mitochondrial calcium uniporter (MCU) and other MCU-regulatory proteins, such as MCUR1 (MCU-regulatory protein 1), essential mCU regulator (EMRE) were also performed. C, MCU (upper bands), MCUR1 (second bands), and EMRE (third bands) expressions were normalized to a-tubulin (lower bands). D through F, Bar graphs show the quantification of protein expression. *P<0.05, vs WT. n=6. KO indicates knockout; PCR, polymerase chain reaction; Sp1, specificity protein; WT, wild-type.

Fig 3: EMRE redistributes in a MCU-dependent manner to the IBM under ER Ca2+ release. a Representative SIM images of HeLa cells that were transiently transfected with EMRE-mCherry (magenta) and MICU1-YFP (green) under resting conditions and 90 s after stimulation with 100 µM histamine. b The sub-mitochondrial localization of EMRE-mCherry under resting conditions (-Hist) and 90 s after treatment with histamine (100 µM) (+Hist) was determined by the IBM association index (n = 8). c Identical experiments as those in a but using cells that overexpressed wild-type MCU. d Identical experiments as those in b but using cells that overexpressed wild-type MCU (n = 8). Horizontal lines represent the median, the lower and upper hinge show respectively first quartile and third quartile, and lower and upper whisker encompass minimal and maximal values. Images and analyses were obtained from at least 10 cells in each of 8 experiments. *P < 0.05 vs. respective control conditions carried out with unpaired double-sided T-test. Source data are provided as a Source Data file

Fig 4: OPA1 and MICU1 tighten the CJ to restrict cytochrome c release from the cristae lumen. a The upper panel shows a single TEM image of a mitochondrion of HeLa cells transfected with control-siRNA (Control si). To analyze the topology of the cristae junction the cristae width was measured in 2 nm increments starting at the thought prolongation of the IBM and measured into the cristae as indicated with red boxes and arrows in a–c. In the lower panel the distance of the CM to the cristae center is plotted against the distance from the IBM (n = 2/24/117 with preparations/cells/CJ). b Same experiment as in a but with OPA1-siRNA (OPA1si) (n = 2/22/97 with preparations/cells/CJ). c Same experiment as in a but with MICU1-siRNA (MICU1si) (n = 2/23/106 with preparations/cells/CJ). d Comparative analysis of the cristae junction topology of Control (a, black), MICU1 (c, magenta) and OPA1 (b, blue) siRNA transfected cells. Data points represent the mean ± SEM (nControl si = 117; nOPA1 si = 97; nMICU1 si = 106). e Representative images of HeLa cells transfected with Control, OPA1, or MICU1 siRNA, stained with MTRCMX, PFA-fixed and immunostained for cytochrome c. f Quantitative analysis of the sub-mitochondrial distribution of cytochrome c in HeLa cells transfected with Control, OPA1, or MICU1 siRNA (n = 12). Horizontal lines represent the median, the lower and upper hinge show respectively first quartile and third quartile, and lower and upper whisker encompass minimal and maximal values. g Schematic representation of the impact of the loss of MICU1 or OPA1 on mitochondrial cytochrome c redistribution caused by enlarged cristae junction. *P < 0.05 vs. respective control conditions carried out with analysis of variance (ANOVA) with Bonferroni post hoc test. Source data are provided as a Source Data file

Fig 5: MICU1 expression is necessary for AT2-to-AT1 differentiation during alveolar epithelial repair and regeneration after SpT4 infection–induced injury.(A) Time line of tamoxifen treatment, SpT4 infection, and lung harvest from adult mice. (B) Confocal images of lung sections at 14 dpi with antibodies to GFP (green) and T1a (red). GFP antibody was used to detect EYFP+ cells. The cell nucleus was stained with DAPI (blue). Arrows point to regions double positive for GFP and T1a. Scale bar: 10 µm. (C) Quantification of the percentage of GFP+/T1a+ area of total GFP+ area per field using ImageJ. (D) Flow cytometry analysis of dissociated lung cells showing the percentage of EYFP+/T1a+ cells from the total of EYFP+ cells at 14 dpi. (E) Pulmonary functions (namely, FVC and FEV0.05) measured at 14 dpi. Data are presented as mean ± SEM. P values were calculated using Student’s t test. **P < 0.01; ***P < 0.001.