Nerve injury-induced protein 2 preserves lysosomal membrane integrity to suppress ferroptosis
- Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, United States
Figures
Figure 1
Nerve injury-induced protein 2 (NINJ2) protein localizes to lysosomes and interacts with lysosome-associated membrane protein 1 (LAMP1).
(A) MCF7 cells were transiently transfected with a plasmid expressing Flag-tagged NINJ2, followed by immunostaining with DAPI, LysoDye and Flag. The arrow indicates co-staining of NINJ2 and LysoDye. (B) MCF7 cells were transiently transfected with a plasmid expressing Flag-tagged NINJ2, followed by immunostaining with anti-Flag and anti-LAMP1. (C–D) 293T cells were transiently transfected with Flag-tagged NINJ2 plasmid for 24 hr, followed by immunoprecipitation with IgG, anti-Flag (C) or anti-LAMP1 (D). The immunocomplex was examined by Western blot analysis with Flag or LAMP1 antibody. (E) MCF7 cells were transfected with Flag-tagged NINJ2 antibody, followed by proximity ligation assay (PLA) assay as described in ‘Materials and methods.’ The positive PLA signal is shown in red puncta.
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Figure 1—source data 1
Original gel images with labels.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig1-data1-v1.zip
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Figure 1—source data 2
Original gels.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig1-data2-v1.zip
Figure 2 with 1 supplement
Loss of Nerve injury-induced protein 2 (NINJ2) leads to enhanced lysosome-associated membrane protein 1 (LAMP1) expression and lysosomal membrane permeability.
(A) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with L-leucyl-L-leucine methyl ester (LLOMe) (1 μM) for 5 hr, followed by immunostaining with antibodies against Galectin 3 and LAMP1. Scale bar: 20 μM. (B–C) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with LLOMe (B) or glucose oxidase (GO) (C) for 5 hr. The cell lysates were subjected to Western blot analysis using antibodies against NINJ2, LAMP1, and actin. The relative protein level of LAMP1 in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (D–E) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with LLOMe (D) or GO (E) for 5 hr, followed by Western blot analysis to detect expression of LAMP1 and actin. The relative protein level of LAMP1 in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane.
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Figure 2—source data 1
Orignal gel images with lableing.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig2-data1-v1.zip
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Figure 2—source data 2
Original gel images.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig2-data2-v1.zip
Figure 2—figure supplement 1
The level of lysosome-associated membrane protein 1 (LAMP1) transcript is increased by loss of Nerve injury-induced protein 1 2 (NINJ2).
The levels of LAMP1 and actin transcripts were measured in isogenic control and NINJ2-KO Molt4 (Left panel) and MCF7 (Right panel) treated with or without 0.25 μM L-leucyl-L-leucine methyl ester (LLOMe) for 5 hr.
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Figure 2—figure supplement 1—source data 1
Original gels with labeling.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original gels.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig2-figsupp1-data2-v1.zip
Figure 3
Loss of Nerve injury-induced protein 2 (NINJ2) leads to increased level of labile iron and reduced expression of ferritin.
(A–B) Isogenic control and NINJ2-KO Molt4 (A) and MCF7 (B) cells were treated with Ferric Ammonium Citrate (FAC) (30 mg/ml) for 24 hr. Cell lysates were collected and the level of labile iron was measured using the QuantiChrom assay kit. * Indicated p<0.05 by Student’s t-test. (C–D) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with FAC (30 mg/ml) for 16 hr, followed by Western blot analysis to measure the level of NINJ2 (C), ferritin light chain (FTL) (C), ferritin heavy chain (FTH) (D), and actin (C–D). The relative protein levels of FTL (C) and FTH (D) in control cells were arbitrarily set as 1.0 and the relative fold change was shown below each lane. (E–F) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with FAC (30 mg/ml) for 16 hr, followed by Western blot analysis to measure the level of FTL (E), FTH (F), and actin (E–F). The relative protein levels of FTL (E) and FTH (F) in control cells were arbitrarily set as 1.0 and the relative fold change was shown below each lane.
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Figure 3—source data 1
Original gels with labeling.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig3-data1-v1.zip
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Figure 3—source data 2
Original gel images.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig3-data2-v1.zip
Figure 4
Loss of Nerve injury-induced protein 2 (NINJ2) promotes ferritin degradation.
(A) The level of ferritin heavy chain (FTH) and HPRT transcripts was measured in isogenic control and NINJ2-KO Molt4 cells. The relative level of FTH transcripts in control cells was arbitrarily set as 1.0 and the relative fold change of transcripts was shown below each lane. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with cycloheximide (50 μg/mL) for 3 to 15 hr. The cell lysates were collected and subjected to Western blot analysis using FTH and actin antibodies. (C) The levels of FTH and actin protein in (B) were quantified and the relative protein half-life of FTH was calculated using GraphPad Prism software. (D) Isogenic control and NINJ2-KO MCF7 cells were transiently transfected with scrambled siRNA or siRNAs against LAMP1 for 3 days, followed by Western blot analysis with antibodies against lysosome-associated membrane protein 1 (LAMP1), FTH, ferritin light chain (FTL), and actin. The relative level of LAMP1, FTH, and FTL protein in control cells was arbitrarily set as 1.0 and the relative fold change of transcripts was shown below each lane.
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Figure 4—source data 1
Original gels with labels.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig4-data1-v1.zip
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Figure 4—source data 2
Original data.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig4-data2-v1.zip
Figure 5
Loss of Nerve injury-induced protein 2 (NINJ2) promotes ferroptosis.
(A) Isogenic control and NINJ2-KO Molt4 cells were treated with RSL3 (1.25 μM) or Erastin (2.5 μM) for 8 hr, and the levels of NINJ2, ferritin heavy chain (FTH), and actin were measured by Western blot analysis. The relative protein level of FTH in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 (1.25 μM) for 8 hr, followed by Western blot analysis to detect FTH and actin. The relative protein level of FTH in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (C–D) Isogenic control and NINJ2-KO Molt4 cells were treated with RSL3 (C) or Erastin (D) from 0 to 7.29 μM for 48 hr. The relative cell viability was measured using the CellTiter-Glo Viability Assay kit. The relative cell viability is calculated as a percentage of untreated (control) cells. Data points represent the mean ± SD from four representative experiments. Curves were fitted using nonlinear regression in GraphPad Prism Software. IC50 values represent the drug concentration required to achieve 50% inhibition of maximal proliferation capacity. (E–F) Isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 (E) or Erastin (F) from 0 to 7.29 μM for 48 hr, followed by cell viability assay. The IC50 was calculated with nonlinear regression analysis using GraphPad Prism Software. (G–H) Colony formation was performed with isogenic control and NINJ2-KO MCF7 cells were treated with or without RSL3 or Erastin for 24 hr. The drugs were then withdrawn to allow colonies to grow for 3 weeks.
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Figure 5—source data 1
Original data with labeling.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig5-data1-v1.zip
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Figure 5—source data 2
Original data.
- https://cdn.elifesciences.org/articles/110919/elife-110919-fig5-data2-v1.zip
Figure 6
Nerve injury-induced protein 2 (NINJ2) and ferritins are overexpressed in hepatocellular and breast carcinomas and are positively associated with one another.
(A–C) Boxplot shows the relative expression of NINJ2 (A), ferritin heavy chain (FTH) (B), and ferritin light chain (FTL) (C) in normal and hepatocellular carcinomas. The analysis was performed using UACLAN database. (D–F) Boxplot shows the relative expression of NINJ2 (A), FTH (B), and FTL (C) in normal and breast carcinomas. The analysis was performed using UACLAN database. (G) NINJ2 expression is positively associated with FTH (G) in hepatocellular carcinomas. The analysis was performed using the GEPIA2 database (http://gepia2.cancer-pku.cn/#correlation). Statistical analysis suggests a strong correlation between NINJ2 and FTH expression in hepatocellular carcinomas (Pearson’s r=0.54). (H) NINJ2 expression is positively associated with FTL (H) in hepatocellular carcinomas (Pearson’s r=0.54). (I–J) NINJ2 expression is positively associated with FTH (I, Pearson’s r=0.54) and FTL (J, Pearson’s r=0.67) in breast carcinomas.
Figure 7
A model to elucidate the role of Nerve injury-induced protein 2 (NINJ2) in maintaining lysosomal membrane integrity and iron homeostasis.
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Nerve injury-induced protein 2 preserves lysosomal membrane integrity to suppress ferroptosis
eLife 15:RP110919.
https://doi.org/10.7554/eLife.110919.3
