pebb flag gcn5  (Addgene inc)

Journal: Cell reports

Article Title: Acetylation-dependent regulation of BRAF oncogenic function

doi: 10.1016/j.celrep.2021.110250

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Flag-GCN5 plasmid , Addgene , Cat#74784.

Techniques: Virus, Recombinant, Cell Fractionation, Mutagenesis, Western Blot, Functional Assay, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Caspase-10 inhibits ATP-citrate lyase-mediated metabolic and epigenetic reprogramming to suppress tumorigenesis

doi: 10.1038/s41467-019-12194-6

Figure Lengend Snippet: Caspase-10 represses proliferative and metastatic genes a A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd), caspase-10/p300 double knockdown (CASP10kd/p300kd) and caspase-10/PCAF double knockdown (CASP10kd/PCAFkd) cells were subjected to glucose starvation for 48 h. The cells were then harvested and sandwich ELISA was performed for acetylated H3 and acetylated H4. Error bars are means ± SD of biological replicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). ** P < 0.01; *** P < 0.001. b A549 control (A549), caspase-10 knockdown (A549 CASP10kd) and caspase-10/GCN5 double knockdown (A549 CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. These cells were harvested and western blotting was performed for the indicated proteins. c A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd) and caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. Total RNA was then isolated and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Heat map comparing relative mRNA levels (mean fold change from three independent experiments with triplicate samples) is shown. Blue and red indicate upregulation or downregulation respectively. d A549 control (A549), caspase-10 knockdown (A549 CASP10kd), caspase-10/ACLY double knockdown (A549 CASP10kd/ACLYkd) and caspase-10/GCN5 double knockdown (A549 CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. ChIP assay was then performed with control IgG or acetylated-H3K9 antibody. Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file

Article Snippet: FLAG-tagged and Myc-tagged caspase-10 (pCMV6-CASP10D) and FLAG-tagged ACLY (pCMV6-ACLY) constructs were purchased from Origene. pEBB Flag GCN5 was a gift from Ezra Burstein (Addgene plasmid # 74784). pAd-Track Flag GCN5 Y621A/P622A was a gift from Pere Puigserver (Addgene plasmid # 14425).

Techniques: Control, Knockdown, Sandwich ELISA, Western Blot, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Caspase-10 inhibits ATP-citrate lyase-mediated metabolic and epigenetic reprogramming to suppress tumorigenesis

doi: 10.1038/s41467-019-12194-6

Figure Lengend Snippet: Caspase-10 represses ACLY promoted malignant phenotype a , b A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd), caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd), and caspase-10/ACLY/GCN5 triple knockdown (CASP10kd/ACLYkd/GCN5kd) cells were subjected to glucose starvation for 48 h. The cells were treated with sodium acetate (5 mM), palmitic acid (0.2 mM) or both for the last 24 h of starvation period as indicated. a In vitro invasion, and ( b ) migration potential of the cells was then measured. Error bars are means ± SD of three biological replicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). *** P < 0.001. c A549 control, A549 CASP10kd, A549 CASP10kd/ACLYkd, A549 CASP10kd/GCN5kd, and A549 CASP10kd/ACLYkd/GCN5kd cells were seeded in soft agar and maintained in glucose starvation (5 mM) conditions, and treated with sodium acetate (5 mM), palmitic acid (0.2 mM) or both as indicated. Scale bars: 200μm. Representative images of colonies are shown. d A549 control (control), A549 CASP10kd, A549 CASP10kd/ACLYkd, and A549 CASP10kd/GCN5kd cells were injected subcutaneously into nude mice. Post-one week of injection, mice were treated with metformin (5 mg/ml in drinking water). Tumor volume was measured on the indicated days. The data shown are representative of three biological replicates ( n = 5 mice/independent experiment). Error bars represent mean ± SD from five individual mice. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). **** P < 0.0001. e At the end of 30 days, tumors were excised and weighed. The data shown are representative of three biological replicates ( n = 5 mice/independent experiment). Error bars represent mean ± SD from five individual mice. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). * P < 0.05; *** P < 0.001. f Immunofluorescence staining for Ki67 was performed on tumor sections (panel d above). Results shown is the representative of staining in five individual biological samples performed in triplicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar: 10 μm. g At the end of 30 days, lysates of tumors (panel d above) were analyzed by immunoblotting. The data shown are representative of three independent experiments. Source data are provided as a Source Data file

Article Snippet: FLAG-tagged and Myc-tagged caspase-10 (pCMV6-CASP10D) and FLAG-tagged ACLY (pCMV6-ACLY) constructs were purchased from Origene. pEBB Flag GCN5 was a gift from Ezra Burstein (Addgene plasmid # 74784). pAd-Track Flag GCN5 Y621A/P622A was a gift from Pere Puigserver (Addgene plasmid # 14425).

Techniques: Control, Knockdown, In Vitro, Migration, Injection, Immunofluorescence, Staining, Western Blot

Journal: Nature Communications

Article Title: Caspase-10 inhibits ATP-citrate lyase-mediated metabolic and epigenetic reprogramming to suppress tumorigenesis

doi: 10.1038/s41467-019-12194-6

Figure Lengend Snippet: Caspase-10 downregulates ACLY-promoted aggressive tumor phenotype a A549 Luc2 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd), and caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd) cells were orthotopically injected into the lung of nude mice. Post-one week of injection, mice were administered metformin (5 mg/ml in drinking water). Bioluminescence imaging was performed weekly and representative images are shown. The data shown are representative of three independent experiments using five individual mice per group. b Bioluminescence quantification (panel a above) was performed at indicated time points. The data shown are representative of three independent experiments using five individual mice per group. Error bars are mean ± SD from five individual mice (n = 5 mice per group). Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). *** P < 0.001. c At the end of 4 weeks, blood from mice in (panel a above), was used to isolate genomic DNA for examining circulating tumor cells. The data shown are representative of three independent experiments using five individual mice per group. Error bars are mean ± SD from five individual mice ( n = 5 mice per group). Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). *** P < 0.001. d At the end of 5 weeks, tumor lysates were prepared (panel a above) and analyzed by immunoblotting for the indicated proteins. The data shown are representative of three independent experiments using five individual mice per group. e Representative imag e of immunostaining of ACLY, caspase-10 and H3K9Ac in different grades of human lung adenocarcinoma and matched normal adjacent tissue (NAT). DAPI was used to counter stain nucleus. Scale bar: 20 μm. f Quantitation of ACLY, caspase-10 and H3K9Ac levels in different grades of human lung adenocarcinoma with respect to matched normal adjacent tissue. The data shown are representative of three independent experiments. Error bars are mean ± SD. Statistical analyses were done using one-way ANOVA (Dunn’s multiple comparison test). * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file

Article Snippet: FLAG-tagged and Myc-tagged caspase-10 (pCMV6-CASP10D) and FLAG-tagged ACLY (pCMV6-ACLY) constructs were purchased from Origene. pEBB Flag GCN5 was a gift from Ezra Burstein (Addgene plasmid # 74784). pAd-Track Flag GCN5 Y621A/P622A was a gift from Pere Puigserver (Addgene plasmid # 14425).

Techniques: Control, Knockdown, Injection, Imaging, Western Blot, Immunostaining, Staining, Quantitation Assay, Comparison