Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: 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).

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: Activity Assay, Confocal Microscopy, Staining, Incubation, Western Blot, Immunoprecipitation, Knockdown, Translocation Assay, Transfection, Fluorescence, Control

Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: 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 H 2 O 2 assay proved the level of H 2 O 2 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).

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: In Vivo, Western Blot, Translocation Assay, Control, Activity Assay, Immunoprecipitation

Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: DPT induces PARP1 over-activation via causing DNA double strand breaks (DSBs). The activated PARP1 promotes TAX1BP1 translocation from cytoplasm to mitochondria by depletion of its substrate NAD + . Then, TAX1BP1 enhances the activation of respiratory chain complex I not only by increasing the protein levels of ND1 and ND2, but also via promoting their assemblies into complex I. The activated respiratory chain complex I generates more superoxide, resulting AIF translocation from mitochondria to nuclei. Within nuclei, AIF serves as a nuclease to cause chromatinolysis to complete the final stage of parthanatos.

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: Activation Assay, Translocation Assay