Fig 2: Mitophagy‐deficient cancer cells are sensitive to ferroptosis inducers. A,F) Immunoblot showing the expression of PINK1 in the indicated HCT116 cells (A) or MDA‐MB‐231 cells (F). B–E) Cell death and lipid peroxidation measurement in the indicated HCT116 cells treated with 12 µm erastin for 26 h (B,C) or cystine deprivation for 19 h (D,E). G–J) Cell death and lipid peroxidation measurement in the indicated MDA‐MB‐231 cells treated with 2 µm erastin for 12 h (G, H) or cystine deprivation for 7 h (I,J). K) Immunoblot showing the expression of Tom20 or Tim23 in HCT116 cells treated with control or mitophagy inbibitor 10 µm Mdivi‐1 for 18 h. (L‐O) Cell death and lipid peroxidation measurement in the indicated HCT116 cells treated with 12 µm erastin for 26 h (L,M) or cystine deprivation for 19 h (N,O) with or or without 10 µm Mdivi‐1 or 10 µm Lipro‐1. P) Immunoblot showing the expression of Tom20 or Tim23 in MDA‐MB‐231 cells treated with control or 10 µm Mdivi‐1 for 12 h. Q–T) Cell death and lipid peroxidation measurement in the indicated MDA‐MB‐231 cells treated with 2 µm erastin for 12 h (Q, R) or cystine deprivation for 7 h (S,T) with or or without 10 µm Mdivi‐1 or 10 µm Lipro‐1. (A,F,K,P) Data are representative of n = 3 biologically independent experiments. (B–E,G–J,L–O,Q–T, Data are the mean ± s.d.; n = 3 biologically independent experiments. Statistical analysis was performed using an unpaired two‐tailed Student's t‐test.

Fig 3: Identification of caspase-2-downstream proteins regulating mitochondria-related gene transcription. A Protein–protein interaction network of the predicted transcription factors and caspase-2, based on the STRING database. B Representative immunoblots and relative protein levels of CASP2 and p53 in SW620 WT and SW620 Casp2−/− cells. C Relative gene expression of Tp53, Sdhb, Timm21, Timm23, Tomm20, and Tomm40 in SW620 Casp2.−/− cells transfected with siRNA targeting Tp53 or negative control (NC). D Representative immunoblots and (E) relative protein levels of CASP2, p53, SDHA, SDHB, TIMM23, TOMM20, and TOMM40 in SW620 Casp2-KO and SW620 Casp2-KO + shp53 cells. Results are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: Effects of caspase-2 on mitochondria-related gene expression and protein content in colorectal adenocarcinoma cells. A Representative immunoblots and relative protein levels of CASP2, SDHA, SDHB, TIMM21, TIMM23, TOMM20, and TOMM40 in SW620 WT and SW620 Casp2−/− cells. B Representative immunoblots and relative protein levels of CASP2, SDHB, TIMM21, TIMM23, TOMM20, and TOMM40 in SW620 WT cells transfected with siRNA targeting Casp2 or negative control (NC). C Relative expressions of mitochondria-associated genes: Casp2, Sdha, Sdhb, Timm21, Timm23, Tomm20, and Tomm40 in SW620 WT and SW620 Casp2−/− cells. Results are shown as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant

Fig 5: PLIN5 localize on the outer mitochondrial membrane in BAT(a) Western blot for PLIN5, Actin, TIM23 and TOM20 from whole cell lysate and pure mitochondria with and without treatment with Proteinase K from control or BKOPLIN5 mice housed at 23 or 6 °C for 16 hours.