Fig 2: NAD+ depletion promoted TAX1BP1 accumulation on mitochondria.a and b NAD+ assay showed the NAD+ depletion caused by 450 nmol/L DPT at 24 h was significantly prevented by prior incubation with 500 μmol/L 3AB for 1 h, but aggregated by pretreatment with 100 μmol/L FK866 for 1 h. c LDH release assay demonstrated that the glioma cell death induced by 450 nmol/L DPT at 24 h was significantly prevented by pretreatment with exogenous NAD+ at 2 mmol/L for 1 h, but aggregated by incubation with 200 μmol/L FK866 for 1 h. d Western blotting showed that incubation with exogenous 2 mmol/L NAD+ for 1 h obviously suppressed the upregulation of TAX1BP1, ND1 and ND2 in mitochondrial fractions and the downregulation of TAX1BP1 in cytoplasmic fractions caused by 450 nmol/L DPT at 24 h. e Western blotting proved that DPT-induced upregulation of TAX1BP1, ND1 and ND2 in mitochondrial fractions and downregulation of TAX1BP1 in cytoplasmic fractions were all exacerbated by incubation with 200 μmol/L FK866 for 1 h. f Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited in the cells incubated with exogenous 2 mmol/L NAD+ for 1 h, but exacerbated by treatment with 200 μmol/L FK866 for 1 h. g Statistical analysis of the intensity of the red fluorescence detected by MitoSOX red revealed that the increase of mitochondrial superoxide caused by 450 nmol/L DPT at 24 h was inhibited by NAD+, but exacerbated by FK866. The values are expressed as mean ± SD (n = 5 per group).

Fig 3: DPT triggered NAD+ depletion and mitochondrial translocation of TAX1BP1 in vivo.a Representative images of nude mice with xenografted gliomas showed that tumor growth was significantly inhibited when the mice were treated with DPT at a dose of 10 mg/kg for 8 consecutive days. b Statistical analysis of the tumor volumes confirmed that the growth of the tumor in vivo was significantly inhibited by DPT. c Western blotting analysis revealed that DPT promoted mitochondrial translocation of TAX1BP1 and nuclear translocation of AIF. It also induced marked upregulation of ND1 and ND2 and downregulation of mitochondrial GPX4 and catalase in mitochondrial fractions, and obvious upregulation of PARP1, PAR, p-ATM and p-H2AX in nuclear fractions. d NAD+ assay showed that NAD+ was decreased significantly in DPT-treated group when compared with that in control group. e Complex I activity assay showed that DPT treatment resulted in upregulation of complex I activity in oxidation of NADH in vivo. f H2O2 assay proved the level of H2O2 was increased obviously by DPT in vivo. g Immunoprecipitation with an antibody against ND1 revealed that the co-immunoprecipitated ND2, NDUFS2 and NDUFS4 with ND1 were all significantly increased in the DPT-treated group. The values are expressed as mean ± SD (n = 6 per group).

Fig 4: PARP1 regulated DPT-induced mitochondrial translocation of TAX1BP1 in glioma cells.a Western blotting showed that pretreatment with 500 μmol/L 3AB for 1 h obviously prevented the upregulation of nuclear PARP1, PAR, mitochondrial TAX1BP1, ND1, ND2 and downregulation of cytoplasmic TAX1BP1 triggered by 450 nmol/L DPT at 24 h. b Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited in the cells pretreated with 500 μmol/L 3AB for 1 h. c Statistical analysis of the intensity of the red fluorescence detected by MitoSOX red proved that pretreatment with 500 μmol/L 3AB for 1 h obviously prevented the increase of mitochondrial superoxide induced by 450 nmol/L DPT at 24 h. d Western blotting showed that the upregulated PARP1 and PAR in nuclear fractions, TAX1BP1, ND1 and ND2 in mitochondrial fractions and the downregulated TAX1BP1 in cytoplasmic fractions induced by 450 nmol/L DPT at 24 h were all inhibited when PARP1 was knocked down with siRNA. e Complex I activity assay showed that the increased complex I activity caused by 450 nmol/L DPT at 24 h was inhibited by knocking down PARP1 with siRNA. f Statistical analysis of the intensity of the red fluorescence detected by MitoSOX red revealed that knockdown of PARP1 with siRNA significantly prevented the increase of mitochondrial superoxide caused by 450 nmol/L DPT at 24 h. The values are expressed as mean ± SD (n = 5 per group).

Fig 5: TAX1BP1 promoted DPT-induced activation of complex I.a Complex I activity assay showed that 450 nmol/L DPT enhanced complex I activity in oxidation of NADH in a time-dependent manner. b Representative images of confocal microscopy combined with immunochemical staining showed ND1 level was increased obviously in the mitochondria of the cells incubated with 450 nmol/L DPT for 24 h. c Western blotting analysis revealed 450 nmol/L DPT upregulated both ND1 and ND2 in a time-dependent manner. d Co-immunoprecipitation analysis showed that the protein levels of ND2, NDUFS2 and NDUFS4 co-immunoprecipitated with ND1 were increased by 450 nmol/L DPT at 12 h, which became more apparent when incubation time of DPT was increased to 24 h. e Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited by pretreatment with 2 µmol/L rotenone for 1 h. f Western blotting revealed that knockdown of ND1 with siRNA not only apparently inhibited ND1 upregulation in mitochondria, but also alleviated nuclear translocation of AIF induced by 450 nmol/L DPT at 24 h. g Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited in the cells transfected with ND1 siRNA. h Statistical analysis of the intensity of the red fluorescence detected by MitoSOX red revealed that the increased mitochondrial superoxide induced by 450 nmol/L DPT at 24 h was suppressed when ND1 was knocked down with siRNA. i Complex I activity assay showed that knockdown of ND1 with siRNA significantly prevented the increase of complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h. j Western blotting revealed knockdown of TAX1BP1 with siRNA prevented the upregulation of ND1 and ND2 and downregulation of GPX4 and catalase in mitochondrial fractions induced by 450 nmol/L DPT at 24 h. k Co-immunoprecipitation analysis showed that the enhanced interactions between ND1, ND2, NDUFS2 and NDUFS4 caused by 450 nmol/L DPT at 24 h were all inhibited when TAX1BP1 was knocked down with siRNA. *P < 0.01 versus control group. The values are expressed as mean ± SD (n = 5 per group).