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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
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cas9  (Addgene inc)
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using <t>CRISPR/Cas9</t> and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.
Cas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using CRISPR/Cas9 and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.

Journal: Breast Cancer : Targets and Therapy

Article Title: Pharmacological Myristoylation Inhibition by Zelenirstat Disrupts Growth Signalling, Energy Metabolism, and Migration in Triple-Negative Breast Cancer Cells

doi: 10.2147/BCTT.S619042

Figure Lengend Snippet: Impact of NDUFAF4 KO in MDA-MB-231 on various mitochondrial complex I proteins. ( A–C ) NDUFAF4 was knocked out in MDA-MB-231 cells using CRISPR/Cas9 and the resulting cells were examined for the myristoylated mitochondrial complex I assembly factor NDUFAF4 ( A ) as well as non-myristoylated proteins NDUFAF3, NDUFS8, and NDUFB8 ( A and B ), and the myristoylated complex I protein NDUFB7 ( A and C ). Protein levels were normalized to Actin (n≥3). ( D ) Blue native PAGE analysis displaying the total assembled complex I protein level in parental or NDUFAF4 KO MDA-MB-231. *** = p<0.001, **** = p<0.0001. ( E ) Confocal microscopy of COS-7 cells transfected with plasmids expressing either EGFP, WT-NDUFAF4-EGFP, or G2A-NDUFAF4-EGFP cDNAs. Localization of NDUFAF4 was compared with cells both transfected with WT NDUFAF4 and treated with zelenirstat for 48 hours. Transfection of a plasmid expressing pyruvate dehydrogenase (PDH-mCherry) was used to visualize mitochondria. Scale bars represent 5µm.

Article Snippet: EnGen Cas9 NLS and S. pyogenes (NEB) proteins were incubated with prepared gRNAs to form ribonucleoprotein complexes, which were subsequently transfected into MDA-MB-231 cells using Lipofectamine CRISPRMAX according to the manufacturer’s instructions.

Techniques: CRISPR, Blue Native PAGE, Analysis, Confocal, Microscopy, Transfection, Expressing, Plasmid Preparation