(A) Correlation between the onset of clasping and survival. Each point represents data from a single mouse. p=0.0047, Pearson’s test. (B) Age at which Ndufs4−/− mice exhibited the clasping phenotype …
(A) Age at which Ndufs4−/− mice exhibited the clasping phenotype on chow diet. Mice were treated with either vehicle, deferiprone (DFP) in the water (0.5 or 1 mg/mL), or deferoxamine (DFO) via daily …
Complete blood count in PND30 WT or Ndufs4−/− mice on normal (40 ppm) or low (8 ppm) AIN-93G synthetic diet to quantify (A) hematocrit, (B) hemoglobin, (C) red blood cell count which were used to …
Quantification of total iron by ICP-MS from WT and Ndufs4−/− mice at PND35 fed control (40 ppm) or low (8 ppm) AIN-93G in (A) liver, (B) whole brain, (C) kidney, (D) heart, (E) quadricep, (F) …
(A) Left, Box, and whisker plot of combined z-score-normalized ICP-MS values of tissue iron, (B) manganese, (C) zinc, and (D) copper in WT and Ndufs4−/− mice on control (40 ppm) or low (8 ppm) …
(A) Weight at time of tissue collection (PND35) of WT or Ndufs4−/− mice on a normal (40 ppm) or low (8 ppm) AIN-93G synthetic diet. (B) Percent of tissue weight from mice in (A) relative to total …
(A) Quantification of non-heme iron by ferrozine assay and (B) MDA-TBA adduct in livers from WT and Ndufs4−/− mice at PND35 that were fed control (40 ppm) or low (8 ppm) AIN-93G synthetic diet. (C) …
Raw unedited immunoblots for Figure 3D brain regions.
(A) Representative western blot images and (B) densitometry (relative to actin) of proteins involved in regulation of iron transport, storage, or metabolism in livers from PND35 WT and Ndufs4−/− …
Raw unedited immunoblots for Figure 4A liver.
Translation is blocked for 5′-IREs (e.g., Fth1, Ftl1, and Fpn1) when iron regulatory proteins (IRPs) are bound to the IRE in low iron conditions (top left). Protein expression increases due to mRNA …
(A) Representative western blot images and (B) densitometry (relative to actin) of proteins involved in regulation of iron transport, storage, or metabolism in whole brains from PND35 WT and Ndufs4−/…
Raw unedited immunoblot images for Figure 4—figure supplement 2A brain.
Metals were measured as µg metal relative to total dry weight of tissue. N=3–5 mice, - p<0.10, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with post hoc Tukey.
Ndufs4+/+ Mice | Ndufs4−/− Mice | p Value | |||||
---|---|---|---|---|---|---|---|
Metal (µg/g) | AIN-93G normal iron | AIN-93G low iron | AIN-93G normal iron | AIN-93G low iron | WT-Con versus KO-Con | KO-Con versus KO-Low | |
Liver | Fe | 122.9±18.9 | 48.0±3.5 | 352.5±55.5 | 62.5±3.6 | ** | *** |
Mn | 2.20±0.33 | 5.68±0.63 | 3.70±0.44 | 8.62±0.51 | **** | ||
Zn | 47.5±5.07 | 62.0±5.8 | 60.9±5.0 | 71.2±4.0 | |||
Cu | 9.6±0.64 | 12.7±0.9 | 13.9±1.5 | 15.6±1.2 | - | ||
Brain | Fe | 39.3±0.78 | 40.6±2.5 | 39.6±1.8 | 37.8±1.9 | ||
Mn | 1.46±0.04 | 1.94±0.05 | 1.71±0.09 | 1.91±0.09 | |||
Zn | 37.7±0.53 | 48.3±1.6 | 44.1±1.8 | 49.1±3.1 | |||
Cu | 10.3±0.52 | 11.4±0.5 | 11.8±0.4 | 12.1±0.7 | |||
Kidney | Fe | 101.9±6.4 | 77.1±12.4 | 161.5±15.4 | 85.8±3.5 | * | ** |
Mn | 4.54±0.44 | 5.53±0.31 | 4.48±0.20 | 6.01±0.51 | * | ||
Zn | 55.8±1.8 | 56.6±2.6 | 58.9±4.8 | 58.4±5.7 | |||
Cu | 15.1±0.3 | 15.3±0.9 | 17.5±0.7 | 18.0±0.9 | - | ||
Heart | Fe | 226.8±6.9 | 182.7±9.5 | 259.2±26.9 | 230.7±24.0 | ||
Mn | 2.51±0.09 | 3.00±0.05 | 2.59±0.19 | 3.72±0.35 | ** | ||
Zn | 46.6±3.0 | 47.7±1.5 | 31.5±3.0 | 41.3±5.3 | * | ||
Cu | 30.5±1.2 | 30.5±0.1 | 31.7±2.9 | 40.4±2.8 | * | ||
Skeletal Muscle | Fe | 31.2±1.4 | 23.3±2.1 | 35.4±3.9 | 33.0±5.3 | ||
Mn | 0.53±0.05 | 0.83±0.08 | 0.65±0.08 | 0.88±0.10 | |||
Zn | 20.7±0.7 | 21.8±1.4 | 22.5±2.8 | 18.8±1.8 | |||
Cu | 3.80±0.24 | 3.85±0.22 | 4.60±0.56 | 4.40±0.44 | |||
Spleen | Fe | 710.4±59.1 | 316.9±22.3 | 860.5±265.6 | 507.1±43.7 | ||
Mn | 0.94±0.09 | 1.28±0.08 | 0.92±0.40 | 1.45±0.23 | |||
Zn | 110.5±7.5 | 123.1±10.4 | 109.0±38.7 | 126.0±6.8 | |||
Cu | 5.13±0.55 | 6.43±0.43 | 3.84±1.26 | 5.29±0.57 | |||
Duodenum | Fe | 150.1±34.2 | 40.0±8.6 | 243.0±66.3 | 51.7±6.2 | * | |
Mn | 6.78±1.2 | 12.88±2.63 | 6.76±1.28 | 17.1±1.7 | ** | ||
Zn | 89.2±2.2 | 84.9±12.7 | 101.4±9.6 | 114.1±9.3 | |||
Cu | 9.4±0.3 | 8.4±1.3 | 10.8±0.9 | 11.2±0.8 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | Ndufs4tm1.1Rpa C57Bl/6NCrl | Palmiter Laboratory Kruse et al., 2008 | ||
Other | PicoLab Mouse Diet 20 | LabDiet | Cat. 5058 | Facility Chow, Mouse Diet Studies |
Other | AIN-93G Growth Purified Diet | LabDiet | Cat. 57W5 | Normal Iron (40 ppm), Mouse Diet Studies |
Other | AIN-93G Growth Purified Diet | LabDiet | Cat. 5SSU | Low Iron (8 ppm), Mouse Diet Studies |
Chemical compound, drug | Deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone) | Sigma-Aldrich | Cat. 379409 | CAS 30652-11-0 |
Chemical compound, drug | Ferric hydroxide dextran complex | Sigma-Aldrich | Cat. D8517 | CAS 9004-66-4 |
Chemical compound, drug | Trace metal grade concentrated HNO3 | Thermo Fisher Scientific | Cat. A509P500 | CAS 7697-37-2 |
Chemical compound, drug | Low trace metals 30% H2O2 solution | Thermo Fisher Scientific | Cat. NC1199178 | |
Chemical compound, drug | Ultra Trace Elemental Analysis Grade H2O | Thermo Fisher Scientific | Cat. W9-500 | |
Chemical compound, drug | Ferrozine iron reagent, hydrate, 98% pure | Thermo Fisher Scientific | Cat. AC410570010 | CAS 1266615-85-3 |
Chemical compound, drug | Trichloroacetic acid, 99% | Thermo Fisher Scientific | Cat. AAA1115636 | CAS 76-03-9 |
Chemical compound, drug | Thioglycolic acid | Thermo Fisher Scientific | Cat. AAB2039122 | CAS 68-11-1 |
Chemical compound, drug | HALT Protease and Phosphatase Inhibitor Cocktail (100X) | Thermo Fisher Scientific | Cat. 78444 | |
Other | Bovine Serum Albumin, Heat Shock Treated | Thermo Fisher Scientific | BP1600-100 | Western Blot Assays |
Chemical compound, drug | RIPA Lysis Buffer | Thermo Fisher Scientific | Cat. 89901 | |
Chemical compound, drug | RestorePlus Stripping Buffer | Thermo Fisher Scientific | Cat. 46430 | |
Commercial assay or kit | TBARS Assay Kit | Cayman Chemical | Cat. 10009055 | |
Commercial assay or kit | SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Fisher Scientific | Cat. 34578 | |
Commercial assay or kit | SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Fisher Scientific | Cat. 34095 | |
Commercial assay or kit | Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | Cat. 23225 | |
Commercial assay or kit | PureLink RNA Mini Kit | Thermo Fisher Scientific | Cat. 12183025 | |
Commercial assay or kit | iTaq Universal SYBR Green One-Step Kit | Bio-Rad | Cat. 1725151 | |
Commercial assay or kit | Phire Tissue Direct PCR Master Mix | Thermo Fisher Scientific | Cat. F170L | Genotyping |
Commercial assay or kit | No-Stain Protein Labeling Reagent | Thermo Fisher Scientific | Cat. A44717 | |
Antibody | Anti-FTH1 (rabbit polyclonal) | Cell Signaling Technology | Cat. cs-3998 | 1:5000 |
Antibody | Anti-TFR1 (rabbit monoclonal) | Abcam | Cat. ab214039 | 1:3000 |
Antibody | Anti-FPN1 (rabbit polyclonal) | Thermo Fisher Scientific | Cat. PA5-77470 | 1:3000 |
Antibody | Anti-DMT1 (mouse monoclonal) | Santa Cruz Biotechnology | Cat. sc-166884 | 1:3000 |
Antibody | Anti-IRP1 (rabbit monoclonal) | Cell Signaling Technology | Cat. cs-20272 | 1:1000 |
Antibody | Anti-IRP2 (rabbit monoclonal) | Cell Signaling Technology | Cat. cs-37135 | 1:1000 in 1% BSA |
Antibody | Anti-GFAP (rabbit monoclonal) | Cell Signaling Technology | Cat. cs-12389 | 1:5000 |
Antibody | Anti-Actin HRP conjugate (rabbit monoclonal) | Cell Signaling Technology | Cat. cs-5125 | 1:5000 |
Antibody | Anti-rabbit IgG (H+L) secondary antibody, HRP (donkey polyclonal) | Thermo Fisher Scientific | Cat. 31458 | 1:20,000 |
Other | m-IgGκ binding protein HRP conjugate | Santa Cruz Biotechnology | Cat. sc-516102 | 1:2000, Western Blot Assays |
Sequence-based reagent, primer | Forward: TCAAGCCAGATCAGCATTCTC Reverse: AGCCAGTTTCATCTCCACATG | Integrated DNA Technologies | Tfr1 | |
Sequence-based reagent, primer | Forward: TCCTCATCACCATCGCAGACACTT Reverse: TCCAAACGTGAGGGCCATGATAGT | Integrated DNA Technologies | Dmt1A/B+IRE Dmt1A/B-IRE | |
Sequence-based reagent, primer | Forward: TGGATGGGTCCTTACTGTCTGCTAC Reverse: TGCTAATCTGCTCCTGTTTTCTCC | Integrated DNA Technologies | Fpn1 | |
Sequence-based reagent, primer | Forward: CTCATGAGGAGAGGGAGCAT Reverse: GTGCACACTCCATTGCATTC | Integrated DNA Technologies | Fth1 | |
Sequence-based reagent, primer | Forward: GTCCCGTGGATCTGTGTCT Reverse: AGGAGCTAACCGCGAAGAGA | Integrated DNA Technologies | Ftl1 | |
Sequence-based reagent, primer | Forward: AAGCAGGGCAGACATTGCGAT Reverse: CAGGATGTGGCTCTAGGCTATGT | Integrated DNA Technologies | Hamp | |
Sequence-based reagent, primer | Forward: GTGTGAACGGATTTGGCCGTATTGGGCG Reverse: TCGCTCCTGGAAGATGGTGATGGGC | Integrated DNA Technologies | Gapdh | |
Sequence-based reagent, primer | Forward: GCTGAGAGGGAAATCGTGCGTG Reverse: CCAGGGAGGAAGAGGATGCGG | Integrated DNA Technologies | Actb | |
Other | Blood collection tube | Thermo Fisher Scientific | Cat. 02-669-33 | Lavendar Cap, CBC Assay |
Other | Metal-Free Centrifuge Tube, 15 mL | VWR | Cat. 89049-172 | ICP-MS Assay |