Fig. 1
Loss of mCa2+ uptake enhances the myofibroblast differentiation. a Mcu conditional allele with LoxP sites flanking exons 5–6. b Experimental timeline for deletion of Mcu in mouse embryonic fibroblasts (MEFs). MEFs were isolated from Mcufl/fl embryos at E13.5 and infected with adenovirus encoding Cre recombinase (Ad-Cre) or the control beta-galactosidase (Ad-βgal) for 24 h. c Expression of mtCU components was examined by Western blot in Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs. MICU1 Mitochondrial Ca2+ Uptake 1, MCUR1 Mitochondrial Ca2+ Uniporter Regulator 1, MCUB Mitochondrial Ca2+ Uniporter B, EMRE Essential MCU Regulator. Mitochondrial loading controls: Voltage-dependent anion channel (VDAC) and complex III (CIII, subunit-UQCRC2) served as mitochondrial loading controls and tubulin as total lysate loading control. d Mcu−/− and control were transduced with adenovirus encoding the mitochondrial calcium sensor, Mito R-GECO. 1 mM ATP was delivered to initiate IP3R-mediated Ca2+ release; n = 13 cells. e Amplitude (peak intensity–baseline). f Mcu−/− and control were loaded with the Ca2+-sensitive dye Fluo-4 AM. Fluorescence was recorded during 1 mM ATP treatment; n = 15 cells Ad-βgal, n = 17 cells Ad-Cre. g Amplitude (peak intensity–baseline). h–l MEFs were treated with TGFβ or AngII for 24 h and immunofluorescence was performed by costaining with α-smooth muscle actin (α-SMA) antibody (red) and DAPI (blue); n = 3. h–j Representative images. k Percentage of α-SMA positive cells. l α-SMA expression (fluorescence intensity). m, n Collagen gel contraction assay; n = 4 Ad-βgal, n = 5 Ad-Cre. m Representative images. n Gel contraction calculated as percent change from time 0 h. o Fold change in expression of myofibroblast genes (vs. Ad-βgal control). Col1a1 collagen type I alpha 1 chain, Col1a2 collagen type I alpha 2 chain, Col3a1 collagen type III alpha 1 chain, α-SMA (Acta2) α-smooth muscle actin, Postn periostin, Lox lysyl oxidase, Fn1 fibronectin 1, Pdgfra platelet derived growth factor receptor alpha col1a1 (n = 7), col1a2 (n = 4), col3a1 (n = 5), aSMA (n = 5), Postn (n = 9), Lox (n = 6), Fn1 (n = 6), Pdgfra (n = 6). p Cell proliferation measured by quantifying DNA content; n = 6. Ca2+ traces: solid line = mean, dashed line = SEM. Data shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle control analyzed by ANOVA. ###p < 0.001, ##p < 0.01, #p < 0.05 vs. Ad-βgal analyzed by t-test. Scale bar = 50 μm. Also see Supplementary Fig. 1
