NINJ2 protein localizes to lysosomes and interacts with 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 costaining 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 hours, followed by immunoprecipitation with IgG, anti-Flag (C) or anti-LAMP1 (D). The immunocomplex were examined by western blot analysis with Flag or LAMP1 antibody. (E) MCF7 cells were transfected with Flag-tagged NINJ2 antibody, followed by PLA assay as described in “Material and Methods”. The positive PLA signal is shown in red puncta.

Loss of NINJ2 leads to enhanced LAMP1 expression and lysosomal membrane permeability

(A) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with LLOMe (1mM) for 5 hours, followed by immunostaining with antibodies against Galectin 3 and LAMP1. Scale bar: 20µM. (B-C) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with LLOMe (B) or GO (C) for 5 hours. The cell lysates were subjected to western blot analysis using antibodies against NINJ2, LAMP1 and actin. (D-E) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with LLOMe (D) or GO (E) for 5 hours, followed by western blot analysis to detect expression of LAMP1 and actin.

Loss of 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 FAC (30mg/ml) for 24 hours. Cell lysates were collected and the level of labile iron was measured using QuantiChrom™ assay kit. (C-D) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with FAC (30mg/ml) for 16 hours, followed by western blot analysis to measure the level of NINJ2 (C), FTL (C), FTH (D) and actin (C-D). (E-F) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with FAC (30mg/ml) for 16 hours, followed by western blot analysis to measure the level of FTL(E), FTH (F) and actin (E-F).

Loss of NINJ2 promotes ferritin degradation

(A) The level of FTH and HPRT transcripts was measured in isogenic control and NINJ2-KO Molt4 cells. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with cycloheximide (50 μg/mL from 3 to 15 hours. The cell lysates were collected and subjected to western blot analysis using FTH and actin antibodies. (C) The level of FTH and actin protein in (B) were quantitated and the relative protein half-life of FTH was calculated by 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 LAMP1, FTH, FTL, and actin.

Loss of 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 hours, and the level of Ninj2, FTH, and actin were measured by western blot analysis. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 (1.25 µM) for 8 hours, followed by western blot analysis to detect FTH, and actin. (C-D) Isogenic control and NINJ2-KO Molt4 cells were treated with RSL3 (C) or Erastin (D) from 0-7.29 µM for 48 hours. The relative cell viability was measured using 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 using 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 0-7.29 µM for 48 hours, 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 hours. The drugs were then withdrawn to allow colonies to grow for 3 weeks.

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), FTH (B), and 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.