EMBO/SourceData huggingface dataset:
Figure 6(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.
Figure 7(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.
Figure 1Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.
Figure 2A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites.B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.
Figure 3A. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications.B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles.C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.
Figure 4A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFPC. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).
Figure 5A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.
Figure 6A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM.B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny.C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.
Figure 7A. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B)C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.
Figure 8A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast.B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control.C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.
Figure 9A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM.C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.
Figure 10TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.
Figure 11Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.
Figure 2Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs. Ago2-associated miRNA (J, left panel) (mean+/- s.e.m., n=3) and mRNA levels (J, right panel) (mean+/- s.e.m., n=2) were also quantified using qRT-PCR.PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).
Figure 6effect of HA-HuR expression on PP2A protein levelseffect of HA-HuR expression on PP2A RNA levels were scored.Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D).Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to halfvariations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).
Figure 8Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on cellular cytokine level RAW 264.7 cells were infected with Antimony sensitive (LdS-Ag83) and antimony resistant (LdR-BHU569) form of Ld for 24 hours along with uninfected cells kept as control. IL-10 (A) and TNF-α (B) were measured at mRNA level by qRT-PCR after infection (mean+/- s.e.m. and, n=4).Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on on phospho-ERK level. (D). Densitometry analysis was done for p-ERK level by ImageJ and relative values were plotted (C). β-Actin was used as loading control Values are mean+/- s.e.m. and, n=3.Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on phospho-ERK and MDR1 level. p-ERK, ERK and MDR1 level were also measured by western blot (D).Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A. RAW 264.7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot (left panel). PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection (right panel). β-Actin was used as loading control.Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.Effect of HuR over expression on LdR infection in mice liver. Pro-inflammatory cytokines TNF-α (left panel) and IL-1β (right panel) level were measured at mRNA level in liver (H).Effect of HuR over expression on LdR infection in mice liver. Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld (I). Values are mean+/- s.e.m. and, n=6).
Figure 1(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples.(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes. (C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test).  (D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.  (E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel.  (F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.  (G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test).  (H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test).  (I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).
Figure 2 (A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test)  (B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001).  (C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked.  (D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm).  (E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e. (F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).
Figure 3 Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates)  Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test)  (C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice.  (D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test).  Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.