Figure 1: Phenotype of MICU1^−/− mouse and mitochondrial Ca²⁺ uptake in MICU1^−/− MEFs.

(a) Map of the MICU1 gene with exon 3 flanked by two loxP sites and a Neomycin selection cassette flanked by flippase recognition target sites (FRTs) and subsequent recombinations for removal of the Neomycin cassette and the exon 3, leading to the truncation of the MICU1 gene. (b) Representative images of haematoxylin and eosin (H&E) staining of diaphragm, heart and lung from MICU1^+/+ and MICU1^−/− animals at e18.5 show no morphological abnormalities (× 40). (c) Quantification of the neuron count in the nucleus ambiguus and the nucleus facialis in toluidine blue-stained sections of the hindbrain of MICU1^+/+ and MICU1^−/− e18.5 animals. Individual points are averages from different animals. Horizontal lines show mean values. Counting of neurons in the nucleus tractus solitarius that contains a subset of inspiratory neurons was not feasible because its borders were less defined. (d) Immunoblots of MICU1, MICU2, MCU and Hsp70 in MICU1^+/+ and MICU1^−/− MEFs lysates. Relative protein level is displayed in the bar graph; each protein was normalized to Hsp70, and expressed relative to MICU1^+/+ MEFs (mean±s.e.m., n=4, *P<0.05, Student’s t-test). (e) Representative [Ca²⁺]_c time courses of the mitochondrial clearance of a 3 μM or 20 μM CaCl₂ bolus (3Ca or 20Ca) in permeabilized MICU1^+/+ and MICU1^−/− MEFs in the presence of thapsigargin (2 μM) and CGP-37157 (20 μM). (f) [Ca²⁺]_c dose response of the initial mitochondrial uptake (30 s after CaCl₂ addition) of different Ca²⁺ boluses recorded as in e. The CaCl₂ doses added were (in μM) 3, 7, 10, 15 and 20 (n=4 per group). A sigmoidal fit is displayed for each. (g) Double logarithmic plot of the initial rates of Ca²⁺ uptake against the peak [Ca²⁺]_c. Slope of each linear fit is indicated (mean±s.e.m., n=4/group, Student’s t-test).