Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 is differentially expressed during embryonic development. a – c Representative western blot for lysates from brain ( a ), heart ( b ), and liver ( c ) harvested from embryo (E13.5), and neonates (P0, P2, and P4). The western blots were probed with antibody specific for MCU, MICU1, MCUR1, and Tom20. d – f Quantification of relative MCU, MICU1, and MCUR1 protein abundance from a – c . g Representative zoomed microscopic images of the tissue microarray probed with MICU1 antibody. h Quantification of MICU1 expression levels represented as percent MICU1 expression as a function of tissue staining. i Representative western blot for lysates from control fibroblasts (GM942), hiPSCs (SeV3, SeV5, and SV20), and hESCs probed with antibody specific for MCU, MICU1, Tom20, and β-actin. j Quantification of relative MICU1 protein abundance from i . k Quantification of MICU1 mRNA levels in different tissues (brain, liver, and heart) harvested from embryo and neonates (E13.5, P0, P2, and P4). l Quantification of MICU1 mRNA levels in two independent clonal hiPSCs lines (SeV3 and SV20) and one clonal line of SeV3 (SeV5). Data represents Mean ± SEM; ** P < 0.01; *** P < 0.001; n = 3–5 (one-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Western Blot, Microarray, Expressing, Staining

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 is transcriptionally regulated by Foxd1 under hypoxia. a Quantification of MICU1 mRNA abundance in hiPSCs exposed to normoxia (20% O 2 ) and hypoxia (5% O 2 ). b Representative western blot for lysates from control fibroblasts and hiPSCs grown under hypoxic/normoxic conditions and probed with antibody specific for MICU1, MCU, ATP5A, and Tom20. c , d Quantification of relative MICU1 ( c ) and MCU ( d ) protein abundance in hiPSCs grown under hypoxic/normoxic conditions. e Quantification of Foxd1 mRNA abundance in hiPSCs under normoxia/hypoxia. f Representative western blot for lysates from control fibroblasts and hiPSCs grown under normoxic/ hypoxic conditions and probed with antibody specific for Foxd1 and β-actin. g Quantification of relative protein abundance of Foxd1 and MICU1 quantified from f and b . h Quantification of Foxd1 mRNA abundance in various tissues (brain, heart, liver, and lung) harvested from different stages of embryonic development. i Representative western blot for lysates from brain, heart, liver, and lung harvested from embryos/neonates and probed with antibodies specific for MICU1, Foxd1, and β-actin. j Quantification of relative protein abundance of Foxd1 and MICU1 quantified from i . k ChIP-assay was performed in hiPSCs exposed to normoxia and hypoxia. Antibody specific for Foxd1 was used to immunoprecipitate the chromatin and the fold enrichment of micu1 promoter relative to the matched input control was quantified by qRT-PCR. Bar represents Mean ± SEM, ** P < 0.01, * P < 0.05, *** P < 0.001; n = 3–5 (One-Way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: Loss of MICU1 in hiPSCs augment MCU-mediated Ca 2+ uptake and overloads matrix Ca 2+ . An equal number of fibroblasts (GM942), hiPSCs (SeV3), and SeV3 expressing MICU1cells (6 × 10 6 ) were permeabilized with digitonin (40 μM), loaded with JC-1 (800 nM) and Fura-FF (1 μM) and changes in Δ Ψ m and m Ca 2+ uptake was measured using a multi-wavelength excitation dual-wavelength emission spectrofluorimeter. a Mean traces of Δ Ψ m in fibroblasts and hiPSCs. b Quantification of basal Δ Ψ m before addition of an extramitochondrial Ca 2+ bolus. c Mean traces of [Ca 2+ ] out measured in permeabilized fibroblasts and hiPSCs. d Quantification of the rate of m Ca 2+ uptake as a function of decrease in bath Ca 2+ fluorescence after an extramitochondrial Ca 2+ pulse (1 μM). e Mean traces of [Ca 2+ ] out after addition of CCCP. f Quantification of total m Ca 2+ released after the addition of CCCP as a function of increase in bath Ca 2+ fluorescence. Foxd1 knock down restores MICU1 gate keeping effect. g – k SeV3 cells were transiently transfected with control and Foxd1 siRNA and 48 h post transfection, Δ Ψ m and m Ca 2+ uptake was measured simultaneously in permeabilized cells. g Mean traces of Δ Ψ m in control (Neg siRNA) and Foxd1 KD hiPSCs. h Mean traces of [Ca 2+ ] out measured in permeabilized control (Neg siRNA) and Foxd1 KD hiPSCs. i Quantification of basal Δ Ψ m before addition of an extramitochondrial Ca 2+ bolus. j Quantification of the rate of m Ca 2+ uptake as a function of decrease in bath Ca 2+ fluorescence after an extramitochondrial Ca 2+ pulse (1 μM). k Quantification of total m Ca 2+ released after the addition of CCCP. Data represents Mean ± SEM; ** P < 0.01, *** P < 0.001; n = 4–6 (One-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Expressing, Fluorescence, Transfection

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: Reconstitution of MICU1 in hiPSCs establishes i Ca 2+ transients and modulates lineage-specific cell differentiation. a Representative western blot for lysates from hiPSCs with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, ATP5A, Cytochrome C, Foxd1, Tom20, and β-actin. b Quantification of relative protein abundance of Foxd1 and MICU1. c Traces of spontaneous cytosolic Ca 2+ oscillations in control fibroblasts and hiPSCs with and without MICU1 expression. Intact cells were loaded with fluo4 and spontaneous Ca 2+ oscillations were observed at 488 nm. d Quantification of peak fluo-4 fluorescence. e Quantification of the frequency of c Ca 2+ oscillations. f Representative western blot for lysates from hiPSCs induced for lineage specific differentiation for the specified days with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, Foxd1, and β-actin. g Traces of spontaneous c Ca 2+ oscillations in hiPSCs upon induction of lineage specific differentiation with and without MICU1 expression. Ca 2+ transients were measured at different days of differentiation: Day 3, 4, and 5 for endoderm and mesoderm; Day 5, 6, and 7 for ectoderm. h Quantification of peak fluo-4 fluorescence. i Quantification of the frequency of c Ca 2+ oscillations. Data indicate quantified peak fluorescence/frequency of oscillation. Mean ± SEM is represented in the figures; ** P < 0.01, * P < 0.05, *** P < 0.001; n = 3–4 (One-Way Anova) individual experiments. Dots represents mean data from group of single cells

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Cell Differentiation, Western Blot, Expressing, Fluorescence

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 expression promotes iPSC-Cardiomyocyte maturation. a Representative western blot for lysates from SeV3 and SeV3-derived cardiomyocytes (SeV3-CMs) with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, Foxd1, Tom20, and β-actin. b Quantification of the MICU1 mRNA abundance in SeV3 and day 24 SeV3-CMs. c Traces of spontaneous cytosolic Ca 2+ oscillations in SeV3-CMs with and without MICU1 expression at day 24 and day 46 post-differentiation. d Quantification of peak fluo-4 fluorescence. e Quantification of the frequency of c Ca 2+ oscillations. Data indicate Mean ± SEM; ** P < 0.001, *** P < 0.001; n = 3–4 independent experiments. Dots represents data from group of single cells. f Quantification of mRNA abundance of the early cardiac transcription factor Mef2c in SeV3-CMs with or without MICU1 expression at day 24 of differentiation. g Quantification of mRNA abundance of the cardiac contractility proteins, MYH6, and MYH7 in SeV3-CMs with or without MICU1 expression at day 24 of differentiation. Data indicate Mean ± SEM; * P < 0.05, *** P < 0.001; n = 3–4. h , j Measurement of oxygen consumption rate (OCR) in SeV3-CMs with or without MICU1 expression using glucose (glucose oxidation) ( h ) or carnitine (fatty acid oxidation) ( j ) as substrate (glucose oxidation). After basal OCR measurement, oligomycin (A), FCCP (B), and rotenone + Antimycin A (C) were added as indicated. h , j Representative traces of OCR in SeV3-CMs. i , k Quantification of basal and maximal OCR, and spare capacity in SeV3-CMs. Data indicate Mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.0001; n = 3

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Expressing, Western Blot, Derivative Assay, Fluorescence