Journal: iScience

Article Title: WASH interacts with Ku to regulate DNA double-stranded break repair

doi: 10.1016/j.isci.2021.103676

Figure Lengend Snippet:

Article Snippet: Plasmid: DR-GFP , , Addgene_26475.

Techniques: Recombinant, In Situ, Plasmid Preparation, Expressing, Construct, Software

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: Inhibitory impact of SIRT4(H161Y) on autophagic flux and mitophagy upon CoCl 2 -induced pseudohypoxia. ( A ) HEK293 cells stably expressing eGFP, SIRT4-eGFP, or SIRT4(H161Y)-eGFP were subjected to CoCl 2 -induced hypoxia followed by immunoblot analysis of LC3B-I/II levels (n=10). ( B, C ) Relative quantification of immunoblot signals of LC3B-II and LC3B-I was performed using ImageJ-based densitometric evaluation and β-actin levels as loading control. ( D ) HEK293 cells stably expressing myc-Flag, SIRT4-myc-Flag, or SIRT4(H161Y)-myc-Flag were subjected to CoCl 2 -induced hypoxia followed by flow cytometry-based analysis of autophagic flux using the GFP-LC3-RFP-LC3ΔG probe (n=8). The more the GFP-LC3/RFP-LC3ΔG ratio decreases, the higher is the autophagic flux. ( E ) Analysis of mitophagic activity using the mt-mKEIMA fluorescent protein (n=4). ( F ) Mitochondrial content analysis using the MitoMark Green I probe (n=7). To determine statistical significance, Two-Way ANOVA tests were employed (mean ± S.D.; *p < 0.05; **p < 0.01; ***p < 0.001). MFI: Median fluorescent intensity.

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Stable Transfection, Expressing, Western Blot, Quantitative Proteomics, Control, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: Ectopic expression of SIRT4 or SIRT4(H161Y) leads to upregulation of HDCA6 protein levels and concomitant reduced acetylated α-tubulin (K40). ( A ) Immunoblot and densitometric ( B , C ) analysis of HDAC6 protein levels in untreated and CoCl 2 -treated HEK293 cells expressing SIRT4 or SIRT4(H161Y) (n=5 to 7). Immunoblot ( D ) and densitometric ( E ) analysis of acetylated α-tubulin (K40) protein levels in untreated and CoCl 2 -treated HEK293 cells expressing SIRT4 or SIRT4(H161Y) (n=8). To determine statistical significance, Two-Way ANOVA tests were employed (mean ± S.D.; *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: The HDAC6 inhibitor tubacin fails to rescue LC3B-II levels, autophagic flux, and mitophagy in SIRT4(H161Y) expressing HEK293 cells upon CoCl 2 -induced pseudohypoxia. ( A ) HEK293 cells stably expressing eGFP, SIRT4-eGFP, or SIRT4(H161Y)-eGFP were subjected to CoCl 2 -induced hypoxia in the presence or absence of the HDAC6 inhibitor Tubacin followed by immunoblot analysis of LC3B-I/II levels. ( B ) Relative quantification of immunoblot signals of LC3B-II was performed using ImageJ-based densitometric evaluation and β-actin levels as loading control (n=3). ( C , D ) HEK293 cells stably expressing FLAG, SIRT4-myc-Flag, or SIRT4(H161Y)-myc-Flag were subjected to CoCl 2 -induced hypoxia in the presence or absence of tubacin followed by flow cytometry-based analysis of autophagic flux ( C ; n=6) and mitophagic activity ( D ; n=3). To determine statistical significance, Two-Way ANOVA tests were employed (mean ± S.D.; **p < 0.01; *** p < 0.001). MFI: Median fluorescent intensity.

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Expressing, Stable Transfection, Western Blot, Quantitative Proteomics, Control, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: SIRT4(H161Y) inhibits CoCl 2 -induced conversion of OPA1-L to OPA1-S. ( A ) Immunoblot and densitometric analysis ( B ) of OPA1-L/OPA1-S protein levels in untreated and CoCl 2 -treated HEK293 cells expressing SIRT4 or SIRT4(H161Y) (n=6). ( C ) The OPA1 inhibitor MYLS22 decreases the OPA1-L/OPA1-S protein ratio as quantified by densitometric analysis ( D ; n=3). Cell treatment was performed for 24 hrs. To determine statistical significance, Two-Way ANOVA tests were employed (mean ± S.D.; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: The OPA1 inhibitor MYLS22 fails to restore LC3B-II levels, autophagic flux, and mitophagy in SIRT4(H161Y) expressing HEK293 cells upon CoCl 2 -induced pseudohypoxia. ( A ) Immunoblot and ( B ) densitometric analysis of LC3B-II protein levels in untreated and CoCl 2 -treated HEK293 cells expressing SIRT4 or SIRT4(H161Y) in the presence or absence of MYLS22 (n=3). ( C ) HEK293 cells stably expressing myc-Flag, SIRT4-myc-Flag, or SIRT4(H161Y)-myc-Flag were subjected to CoCl 2 -induced hypoxia in the presence or absence of MYLS22 followed by flow cytometry-based analysis of autophagic flux using the GFP-LC3-RFP-LC3ΔG probe (n=4). ( D ) HEK293 cells stably expressing eGFP, SIRT4-eGFP, or SIRT4(H161Y)-eGFP were subjected to CoCl 2 -induced hypoxia in the presence or absence of MYLS22 followed by flow cytometry-based analysis of mitophagy using the mt-mKEIMA probe (n=3). To determine statistical significance, One-Way or Two-Way ANOVA tests were employed (mean ± S.D.; *p < 0.05; ***p < 0.001). MFI: Median fluorescent intensity.

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Expressing, Western Blot, Stable Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: BafA 1 treatment does not restore LC3II-B levels in SIRT4(H161Y) expressing HEK293 cells upon CoCl 2 -induced pseudohypoxia. ( A ) Immunoblot analysis and ( B ) densitometric evaluation of LC3B-II vs . β-actin as loading control was performed (n=3). To determine statistical significance, One-Way ANOVA tests were employed (mean ± S.D.; *p < 0.05).

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

doi: 10.1101/2025.04.29.649368

Figure Lengend Snippet: SIRT4(H161Y) increases inhibitory phosphorylation of ULK1 upon CoCl 2 -induced pseudohypoxia. ( A ) HEK293 cells stably expressing eGFP, SIRT4-eGFP, or SIRT4(H161Y)-eGFP were subjected to CoCl 2 -induced hypoxia followed by immunoblot analysis of ULK1 pS758 and ULK1 pS638 levels. ( B ) Relative quantification of immunoblot signals of ULK1 pS758 (upper panel) and ULK1 pS638 (lower panel) was performed using ImageJ-based densitometric evaluation and β-actin levels as loading control (n=3). To determine statistical significance, Two-Way ANOVA tests were employed (mean ± S.D.; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (HEK293: ACC 305).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Quantitative Proteomics, Control