anti prkn parkin  (Cell Signaling Technology Inc)

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone reduces PRKN-mediated BAX degradation and promotes BAX-BAK1 mitochondrial pores formation. (A) WB assays were performed to assess the BAX and BAK1 proteins expression in Huh7 cells treated with ailanthone for 48 h. (B) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression in mitochondria and cytoplasm were measured by WB. (C, D) Huh7 cells were treated with 1.2 μM ailanthone for 48 h. The lysates were immunoprecipitated against BAX, and immunoblotting assays for BAX and BAK1 were performed (C) . The colocalization of BAX and BAK1 was observed (1000×, scale bar: 50 μm). The colocalization area of BAX with BAK1 per cell was quantified (D) . (E) Huh7 cells were treated with MSN-50 (1 μM for 3 h), MSN-50 was then removed, and the cells were treated with 0.6 μM ailanthone for 48 h. CCK8 assay measured cell viability. (F, G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression were measured by WB (F) . The colocalization of BAX and PRKN proteins were observed (1000×, scale bar: 50 μm). The colocalization area of BAX with PRKN per cell was quantified (G) . (H, I) IP analysis of BAX and BAK1 in Huh7 cells treated with 1.2 μM ailanthone for 48 h was conducted. The ubiquitinated modified BAX and BAK1 signals were visualized using a pan-ubiquitin antibody. The quantitative analysis of ubiquitinated BAX and BAK1 proteins expression was normalized to their respective levels in the IP group. Bar, SD. * p < 0.05 or ** p < 0.01.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: Expressing, Immunoprecipitation, Western Blot, CCK-8 Assay, Modification, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone blocks PINK1-PRKN-mediated mitophagy while promoting BAX-BAK1 mitochondrial pores formation and inducing inflammation in vivo . (A, B) IHC detection of LC3, PINK1, PRKN, BAX, BAK1, TNF-α, IL-1β, and IL-6 proteins expression in tumour tissues (100×, scale bar: 500 μm). (C, D) The colocalization of PINK1 with PRKN and BAX with BAK1 in tumour tissues was observed by confocal microscopy imaging (400×, scale bar: 100 μm). The colocalization areas were quantified. Bar, SD. ** p < 0.01.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: In Vivo, Expressing, Confocal Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Schematic representation of ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation. Ailanthone induces mitochondrial damage while blocking PINK1-PRKN-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria in cells, thereby inhibiting hepatocellular carcinoma (HCC) cell proliferation. Furthermore, the inhibition of PRKN protein by ailanthone reduced the ubiquitination and degradation of BAX, promoting the localization of BAX on the outer mitochondrial membrane, forming BAX-BAK1 mitochondrial pores, aggravating the release of mtDNA, and inducing inflammatory responses. Ultimately, the accumulation of damaged mitochondria and the release of inflammatory factors both contribute to the suppressive effect of ailanthone on HCC cell proliferation.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone suppresses mitophagy by disturbing PINK1-PRKN axis. (A, B) WB assays were performed to assess the PINK1 and PRKN proteins expression in Huh7 cells treated with ailanthone (1.2 μM) for different times (A) , and treated with ailanthone for 48 h (B) . (C) Huh7 cells were treated by ailanthone for 48 h, PINK1 and PRKN mRNA levels were detected by qPCR assay. (D, E) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), the proteins expression of PINK1 and PRKN in whole cells (D) , and in mitochondria and cytoplasm (E) were measured by WB assay. (F) Immunoprecipitated against PRKN, and WB assays for PINK1 and PRKN were performed. Quantitative analysis of PINK1 expression normalised to that of PRKN in the IP group. (G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h). The colocalization of PINK1 with PRKN was observed by confocal microscopy (1000×, scale bar: 50 μm). The colocalization area of PINK1 with PRKN per cell was quantified. Bar, SD . * p < 0.05 or ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Expressing, Immunoprecipitation, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone reduces PRKN-mediated BAX degradation and promotes BAX-BAK1 mitochondrial pores formation. (A) WB assays were performed to assess the BAX and BAK1 proteins expression in Huh7 cells treated with ailanthone for 48 h. (B) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression in mitochondria and cytoplasm were measured by WB. (C, D) Huh7 cells were treated with 1.2 μM ailanthone for 48 h. The lysates were immunoprecipitated against BAX, and immunoblotting assays for BAX and BAK1 were performed (C) . The colocalization of BAX and BAK1 was observed (1000×, scale bar: 50 μm). The colocalization area of BAX with BAK1 per cell was quantified (D) . (E) Huh7 cells were treated with MSN-50 (1 μM for 3 h), MSN-50 was then removed, and the cells were treated with 0.6 μM ailanthone for 48 h. CCK8 assay measured cell viability. (F, G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression were measured by WB (F) . The colocalization of BAX and PRKN proteins were observed (1000×, scale bar: 50 μm). The colocalization area of BAX with PRKN per cell was quantified (G) . (H, I) IP analysis of BAX and BAK1 in Huh7 cells treated with 1.2 μM ailanthone for 48 h was conducted. The ubiquitinated modified BAX and BAK1 signals were visualized using a pan-ubiquitin antibody. The quantitative analysis of ubiquitinated BAX and BAK1 proteins expression was normalized to their respective levels in the IP group. Bar, SD. * p < 0.05 or ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Expressing, Immunoprecipitation, Western Blot, CCK-8 Assay, Modification, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone blocks PINK1-PRKN-mediated mitophagy while promoting BAX-BAK1 mitochondrial pores formation and inducing inflammation in vivo . (A, B) IHC detection of LC3, PINK1, PRKN, BAX, BAK1, TNF-α, IL-1β, and IL-6 proteins expression in tumour tissues (100×, scale bar: 500 μm). (C, D) The colocalization of PINK1 with PRKN and BAX with BAK1 in tumour tissues was observed by confocal microscopy imaging (400×, scale bar: 100 μm). The colocalization areas were quantified. Bar, SD. ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: In Vivo, Expressing, Confocal Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Schematic representation of ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation. Ailanthone induces mitochondrial damage while blocking PINK1-PRKN-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria in cells, thereby inhibiting hepatocellular carcinoma (HCC) cell proliferation. Furthermore, the inhibition of PRKN protein by ailanthone reduced the ubiquitination and degradation of BAX, promoting the localization of BAX on the outer mitochondrial membrane, forming BAX-BAK1 mitochondrial pores, aggravating the release of mtDNA, and inducing inflammatory responses. Ultimately, the accumulation of damaged mitochondria and the release of inflammatory factors both contribute to the suppressive effect of ailanthone on HCC cell proliferation.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Membrane