EMBO/SourceData huggingface dataset:
Figure legend: " (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 &lt; 0.0001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (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). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (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). "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P &lt; 0.001, two-tailed paired Student's t-test) "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P &lt; 0.0001). "
NCBI gene IDs:
"OTUD1: 220213
TFRC: 7037"
Figure legend: " Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates) "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P &lt; 0.001, two-tailed unpaired Student's t-test) "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " (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). "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 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. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 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. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 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. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 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. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 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. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " (A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P &lt; 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL/6 mice. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " tumor volume (n=6 biological replicates, mean ± s.e.m., **P &lt; 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P &lt; 0.01, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P &lt; 0.01, ***P &lt; 0.001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 0.05), ***P &lt; 0.0001, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry. "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P &lt; 0.05, **P &lt; 0.01, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 0.05), *P = 0.0119, **P = 0.0015, ***P &lt; 0.001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P &gt; 0.05), **P &lt; 0.01, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P &lt; 0.001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P &lt; 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P &lt; 0.0001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P &lt; 0.0001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 220213"
Figure legend: " (C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P &lt; 0.001, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown. "
NCBI gene IDs:
"OTUD1: 71198"
Figure legend: " (E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " (F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " (H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test). "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: " (I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. "
NCBI gene IDs:
"Otud1: 71198"
Figure legend: "(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm."
NCBI gene IDs:
"aut2: 855498
aut7: 852200
pep4: 855949
prb1: 856649"
Figure legend: "(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997)."
NCBI gene IDs:
"aut2: 855498
aut7: 852200
pep4: 855949
pra1: 855949"
Figure legend: "(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997)."
NCBI gene IDs:
"aut2: 855498
aut7: 852200
pep4: 855949
pra1: 855949"
Figure legend: "Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate."
NCBI gene IDs:
"aut2‐1: 855498
aut2: 855498
aut7: 852200"
Figure legend: "Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate."
NCBI gene IDs:
"aut2: 855498
aut7: 852200"
Figure legend: "(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods)."
NCBI gene IDs:
"GAL4: "
Figure legend: "(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6)."
NCBI gene IDs:
"AUT7: 852200
aut7: 852200
Met25: 851010"
Figure legend: "Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C."
NCBI gene IDs:
"AUT2: 855498
aut2: 855498
AUT7: 852200
aut7: 852200"
Figure legend: "(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm."
NCBI gene IDs:
"aut2: 855498"
Figure legend: "(B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm."
NCBI gene IDs:
"aut7: 852200"
Figure legend: "(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text."
NCBI gene IDs:
"aut2: 855498"
Figure legend: "(a) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy (ICM and DCM) heart failure (HF) patient heart tissue compared with non-failing hearts (NF). Expression levels were normalized to HPRT and GAPDH mRNAs. n=15 per group. Control, non-failing heart."
NCBI gene IDs:
"GAPDH: 
HPRT: 
HACE1: 57531"
Figure legend: "(b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests)."
NCBI gene IDs:
"Gapdh: 
Hprt: 
Hace1: 209462"
Figure legend: "(c-e) Hace1−/− and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation. Echocardiography measurement of (c) LV fractional shortening (FS=(LVEDD−LVESD)/LVEDD × 100%); (d) LV end-diastolic dimension (LVEDD) (mm) and (e) LV end-systolic dimension (LVESD) (mm) are shown. n=6-8 for each group."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(f) Mortality rate of WT and Hace1−/− mice after sTAC is shown by Kaplan-Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period is shown as the ratio of the number of mice survived/total mice analysed for each group. This represents 60% (12/20) for Hace1−/− sTAC mice, 84% (16/19) for Hace1+/+ sTAC mice and 100% (12/12) for both Hace1−/− and Hace1+/+ sham mice on day 4; P value as indicated (log-rank χ2-test)."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(g) Heart weight/body weight ratios (HW/BW) and (h) lung weight/body weight ratio (LW/BW) of WT and Hace1−/− mice 4 days after sTAC or sham surgery. n=15-21 for each group."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(j) Real-time RT-PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene (Nppa), a clinical biomarker for heart hypertrophy, in Hace1−/− sTAC heart; expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group. In all panels, error bars represent s.e.m., one-way analysis of variance were used to calculate P-values."
NCBI gene IDs:
"Gapdh: 
Hprt: 
Hace1: 209462
Nppa: 230899"
Figure legend: "Representative immunohistochemistry staining for (a) ubiquitinated protein (brown); (b) p62 (brown) and (c) GFP-LC3 (brown) in paraffin sections of heart revealing increased accumulation of p62, ubiquitinated protein and LC3 in Hace1−/− sTAChearts. Hearts from three mice in each group were analysed (scale bars, 10 μm)."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(i) Increased Ub proteasome activity in Hace1−/− sTAC hearts as measured by 20S proteasome activity assay (n=4-6 per group). In all panels, error bars represent s.e.m., P value between KO and WT sTAC as indicated (one-way analysis of variance)."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub+ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm."
NCBI gene IDs:
"HACE1: 209462"
Figure legend: "(b) Representative confocal micrographs of Hace1−/− and Hace1+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm."
NCBI gene IDs:
"Hace1: 209462"
Figure legend: "(c,d) WT and Hace1−/− MEF as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P0.001 (one-way analysis of variance). Scale bars, 10 μm."
NCBI gene IDs:
"HACE1: 209462
Hace1: 209462"
Figure legend: "(a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and HACE1Δ ANK) constructs transiently transfected into Hace1−/− MEF for protein aggregate clearance assay. (b-d) Representative confocal micrographs of (b) WT, (c) ΔHECT and (d) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Vehicle-treated MEFs were used as control. Scale bars, 10 μm."
NCBI gene IDs:
"HACE1: 209462"
Figure legend: "(a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with p62 (red) in the perinuclear space on puromycin treatment (8 h) in Hace1−/− NCM transiently expressing HACE1-GFP. p62 was visualized by immunofluorescent staining. Arrows indicate co-localized puncta."
NCBI gene IDs:
"Hace1: 209462
HACE1: 209462"
Figure legend: "(b) Representative confocal images showing HACE1 (red) localizes to LC3+ puncta (green) induced by MG132 treatment (8 h) in Hace1−/−GFP-Lc3Tg NCM cells transiently expressing HACE1-RFP. Arrows indicate co-localized puncta."
NCBI gene IDs:
"Hace1: 209462
Lc3: 67443///66734"
Figure legend: "(c) Primary NCMs from Hace1+/+Lc3Tg and Hace1−/−Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti-GFP antibodies. Gapdh was used as a loading control."
NCBI gene IDs:
"Hace1: 209462
Lc3: 67443///66734"
Figure legend: "(d) Fluorescence microscopy images showing co-localization of HACE1 (green) with Lamp1 (red) on MG132 treatment (8 h) in Hace1−/−NCM cells transiently expressing HACE1-GFP. Lamp1 was visualized by immunofluorescent staining. Scale bar, 10 μm in all images."
NCBI gene IDs:
"Hace1: 209462
HACE1: 209462"