The Ncoa4+/+ and Ncoa4–/– mice were subjected to pressure overload by transverse aortic constriction (TAC) and analyzed 4 weeks after the operation. (A) Representative images of transthoracic M-mode …
Source data for Figure 1.
Physiological and echocardiographic parameters in 8- to 10-week-old Ncoa4+/+ and Ncoa4–/– mice at baseline.
BP, blood pressure; TL, tibia length; LV, left ventricle; LVIDd, end-diastolic left ventricular internal dimension; LVIDs, end-systolic left ventricular internal dimension; IVSd, end-diastolic interventricular septum thickness; LVPWd, end-diastolic left ventricular posterior wall thickness; FS, fractional shortening. The data are the mean ± SEM. n indicates the number of biologically independent samples. The data were evaluated by unpaired, two-tailed Student’s t-test.
Source data for data table provided in Figure 1—source data 2.
(A) Targeted modification of the Ncoa4 gene. Schematic structures of the wild-type genomic Ncoa4 sequence, the targeting construct, the targeted allele, the floxed allele after flippase recognition …
Source data for Figure 1—figure supplement 1.
The Ncoa4+/+ and Ncoa4–/– mice were subjected to TAC. The levels of mRNA were analyzed 4 weeks after the operation (n = 8 biologically independent samples). Gapdh mRNA was used as the loading …
Source data for Figure 1—figure supplement 2.
The Myh6-Cre– and Myh6-Cre mice were subjected to TAC and analyzed 4 weeks after the operation. Echocardiographic parameters of the mice (n = 4 biologically independent samples) are shown. LVIDd and …
Source data for Figure 1—figure supplement 3.
(A) Western blot analysis of FTH1 in Ncoa4+/+ and Ncoa4–/– hearts 4 weeks after TAC. GAPDH was used as the loading control. The right-hand graphs show the densitometric analysis. The average value …
Source data for Figure 2.
Time course of changes in the echocardiographic parameters of left ventricular (LV) fractional shortening (FS), the end-diastolic LV internal dimension (LVIDd) and the end-systolic LV internal …
Source data for Figure 2—figure supplement 1.
(A) Western blot analysis of FTH1 in Ncoa4+/+ and Ncoa4–/– hearts 1 week after transverse aortic constriction (TAC). GAPDH was used as the loading control. The right-hand graphs show the …
Source data for Figure 3.
(A) Tissue levels of total non-heme, ferrous, and ferric iron in Ncoa4+/+ and Ncoa4–/– hearts 4 weeks after transverse aortic constriction (TAC) (n = 6 biologically independent samples). The ratio …
Source data for Figure 4.
Hematological parameters and red cell indices in Ncoa4+/+ and Ncoa4–/– mice.
PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CH, hemoglobin concentration. Data are the mean ± SEM. n indicates the number of biologically independent samples. The data were evaluated by one-way analysis of variance (ANOVA), followed by Tukey–Kramer’s post hoc test. *p=0.0418 and **p=0.0108 versus the corresponding sham-operated group. Exact p-values are provided in Supplementary file 1.
Source data for data table provided in Figure 4—source data 2.
(A) Tissue levels of total non-heme iron in Ncoa4+/+ and Ncoa4–/– livers 4 weeks after TAC (n = 6 biologically independent samples). (B) Serum interleukin 6 (IL-6) levels (n = 8 biologically …
Source data for Figure 4—figure supplement 1.
(A) Homogenates from the heart 4 weeks after TAC were subjected to western blot analysis using antibodies against iron-responsive element-binding protein 2 (IREB2), ferroportin 1 (SLC40A1), and …
Source data for Figure 4—figure supplement 2.
(A) 4-Hydroxy-2-noneal (HNE) staining of heart sections. Scale bar, 50 μm. The right panel shows the quantitative analysis of 4-HNE-positive area to whole heart section (n = 5 biologically …
Source data for Figure 4—figure supplement 3.
(A) The mRNA expression level of Slc7a11 in the heart 4 weeks after TAC (n = 8 biologically independent samples). Gapdh mRNA was used as the loading control. (B) The cystine concentration in the …
Source data for Figure 4—figure supplement 4.
(A) Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …
Source data for Figure 5.
(A) Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …
Source data for Figure 5—figure supplement 1.
Cell death assay. Cell death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit. Isolated mouse cardiomyocytes from Ncoa4+/+ and Ncoa4–/– hearts were treated with the indicated …
Source data for Figure 5—figure supplement 2.
Wild-type C57BL/6J mice were subjected to transverse aortic constriction (TAC) and analyzed 4 weeks after the operation. Ferrostatin-1 (Fer-1) or saline was intraperitoneally administered daily …
Source data for Figure 6.
The wild-type C57BL/6J mice were subjected to TAC. Ferrostatin-1 (Fer-1) was intraperitoneally administered daily starting one day before TAC. (A) The levels of mRNA were analyzed 4 weeks after the …
Source data for Figure 6—figure supplement 1.
The Ncoa4–/– mice were subjected to TAC. Ferrostatin-1 (Fer-1) or saline was intraperitoneally administered daily starting one day before TAC. (A) Echocardiographic parameters of the mice (n = 5 …
Source data for Figure 6—figure supplement 2.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (male Mus musculus) | C57BL/6J | Envigo UK | C57BL/6OlaHsd | |
Strain, strain background (male Mus musculus) | Ncoa4flox/flox;Myh6-Cre+ | This paper | See Materials and methods | |
Strain, strain background (male Mus musculus) | Ncoa4flox/flox;Myh6-Cre– | This paper | See Materials and methods | |
Strain, strain background (male Mus musculus) | Myh6-Cre+ | Nishida et al., 2004 | See Materials and methods | |
Antibody | Mouse monoclonal antibody to NCOA4 | Sigma-Aldrich | SAB1404569, Lot: HC071-1F11, RRID:AB_10759525 | (1:1000) |
Antibody | Rabbit polyclonal antibody to FTH1 | Cell Signaling Technology | 3998, Lot: 2, RRID:AB_1903974 | for western blots, (1:1000); for immunofluorescence, (1:100) |
Antibody | Mouse monoclonal antibody to LC3B | Cell Signaling Technology | 83506, Lot: 1, RRID:AB_2800018 | (1:100) |
Antibody | Rat monoclonal antibody to LAMP2a | Abcam | ab13524, Lot: GR3317907-1, RRID:AB_2134736 | (1:100) |
Antibody | Rat monoclonal antibody to GPX4 | Millipore | MABS1274, Lot: Q2633070, RRID:AB_2885039 | (1:1000) |
Antibody | Rabbit polyclonal antibody to 4-HNE | Millipore | 393207, Lot: 3167038, RRID:AB_566310 | (1:500) |
Antibody | Rabbit polyclonal antibody to IREB2 | Thermo Fisher Scientific | PA1-16543, Lot: TK2666362A, RRID:AB_2126583 | (1:250) |
Antibody | Rabbit polyclonal antibody to SLC40A1 | Alpha Diagnostic International | MTP11-A, Lot: 1169899A3-L, RRID:AB_1619475 | (1:1000) |
Antibody | Mouse monoclonal antibody to TFRC | Thermo Fisher Scientific | 13–6800, Lot: TI275369, RRID:AB_2533029 | (1:1000) |
Antibody | Rabbit polyclonal antibody to SOD2 | Abcam | ab13534, Lot: GR33618-66, RRID:AB_2191667 | (1:1000) |
Antibody | Rabbit polyclonal antibody to HO-1 | Enzo Life Sciences | ADI-SPA-895, Lot: 03301708, RRID:AB_10618757 | (1:1000) |
Antibody | Mouse monoclonal antibody to GAPDH | Sigma-Aldrich | G8795, Lot: 056M4856V, RRID:AB_1078991 | (1:10000) |
Antibody | Mouse immunoglobulin | Santa Cruz biochemistry | sc-2025, RRID:AB_737182 | |
Antibody | Rabbit IgG | Abcam | ab37415, RRID:AB_2631996 | |
Antibody | Fluorescein isothiocyanate-conjugated lectin | Sigma-Aldrich | L4895 | |
Antibody | Alexa Fluor 488 donkey-anti-mouse | Thermo Fisher Scientific | A21202, RRID:AB_141607 | (1:500) |
Antibody | Alexa Fluor 568 donkey-anti-rabbit | Thermo Fisher Scientific | A10042, RRID:AB_2534017 | (1:500) |
Antibody | Alexa Fluor 488 goat-anti-rat | Thermo Fisher Scientific | A11006, RRID:AB_2534074 | (1:500) |
Antibody | IRDye 680LT Donkey anti-Mouse IgG Secondary Antibody | LI-COR Biosciences | 926–68020, RRID:AB_10706161 | (1:10,000) |
Antibody | IRDye 680LT Donkey anti-Rabbit IgG Secondary Antibody | LI-COR Biosciences | 926–68023, RRID:AB_10706167 | (1:10,000) |
Antibody | IRDye 680LT Goat anti-Rat IgG Secondary Antibody | LI-COR Biosciences | 926–68029, RRID:AB_10715073 | (1:10,000) |
Commercial assay or kit | Pierce Protein G Magnetic Beads | Thermo Scientific | 88848 | |
Commercial assay or kit | RNeasy Fibrous Tissue Mini Kit | QIAGEN | 74704 | |
Commercial assay or kit | SuperScript IV First-Strand Synthesis System | Thermo Fisher Scientific | 18091050 | |
Commercial assay or kit | PowerUp SYBR Green Master Mix | Thermo Fisher Scientific | A25742 | |
Commercial assay or kit | Masson's Trichrome Stain Kit | Polysciences, Inc | 25088–1 | |
Commercial assay or kit | VECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG) | Vector Laboratories Inc | PK-6101 | |
Commercial assay or kit | DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine) | Vector Laboratories Inc | SK-4100 | |
Commercial assay or kit | normal donkey serum | Abcam | ab7475, RRID:AB_2885042 | |
Commercial assay or kit | normal goat serum | Abcam | ab7481, RRID:AB_2716553 | |
Commercial assay or kit | ProLong Gold Antifade Reagent with DAPI | Life Technologies | P36935 | |
Commercial assay or kit | Mouse Ferritin ELISA Kit (FTL) | Abcam | ab157713 | |
Commercial assay or kit | Pointe Scientific Iron/TIBC Reagents | Pointe Scientific | 23-666-320 | |
Commercial assay or kit | ELISA Kit for Troponin T Type 2, Cardiac (TNNT2) | Cloud-Clone | SED232Mu | |
Commercial assay or kit | Mouse IL-6 Quantikine ELISA Kit | R and D Systems | M6000B | |
Commercial assay or kit | Iron Assay Kit | Abcam | ab83366 | |
Commercial assay or kit | Lipid Peroxidation (MDA) Assay Kit | Abcam | ab118970 | |
Commercial assay or kit | GSSG/GSH Quantification Kit | Dojindo | G257 | |
Commercial assay or kit | Glutamine Assay Kit | Abcam | ab197011 | |
Commercial assay or kit | Glutamate Assay Kit | Abcam | ab83389 | |
Commercial assay or kit | Electrophoretic Mobility-Shift Assay (EMSA) Kit | Invitrogen | E33075 | |
Commercial assay or kit | Zero Blunt TOPO PCR Cloning Kit | Invitrogen | 451245 | |
Commercial assay or kit | HiScribe T7 Quick High Yield RNA Synthesis Kit | New England Biolabs | E2050S | |
Chemical compound, drug | Ferrostatin-1 | Sigma Aldrich | SML0583 | |
Chemical compound, drug | Isoprenaline hydrochloride | Sigma Aldrich | I5627 | |
Chemical compound, drug | Erastin | Sigma Aldrich | E7781 | |
Chemical compound, drug | 1S,3R-RSL 3 | Sigma Aldrich | SML2234 | |
Chemical compound, drug | calcein-AM | Invitrogen | C1430 | |
Chemical compound, drug | ethidium homodimer-1 | Invitrogen | E1169 | |
Chemical compound, drug | 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) | Invitrogen | D399 | |
Chemical compound, drug | C11-BODIPY | Invitrogen | D3861 | |
Chemical compound, drug | pyridoxal isonicotinoyl hydrazine (PIH) | Abcam | ab145871 | |
Software, algorithm | ImageJ | National Institutes of Health | Version 1.51 r, RRID:SCR_003070 | |
Software, algorithm | GraphPad Prism 8 | GraphPad Software | RRID:SCR_002798 | |
Software, algorithm | EZChrom Elite | Agilent Technologies | Version 3.3.2. |
Quantification and statistical analysis.
The number of independent biological repeats (n) is shown in the figure legends. P values are shown below.