notch1 promoter construct  (Promega)

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Heatmap of known Notch1 target genes and glycolytic genes identified by RNA-seq using A549 cells stably transfected with Notch1 short hairpin RNA (shRNA) or control shRNA. Western blot shows the knockdown of Notch1 expression. b KEGG pathway analysis of genes differentially expressed between A549 cells stably transfected with Notch1 shRNA or control shRNA. c , d The mRNA and protein expression of glycolytic genes in A549 cells stably transfected with Notch1 shRNA or control shRNA were examined by qRT-PCR ( c ) and western blot ( d ) respectively. e ChIP analysis of Notch1 occupancy on promoters of glycolytic genes in A549 cells. IgG: normal serum. The different number after each gene represents the regions containing different Notch1-binding sites. The graph shows the percentage of input. f , g A549 cells were transfected with empty vector (EV), Notch1 intracellular domain (ICD), or TAZ shRNA. Glucose uptake, pyruvate level, lactate production level ( f ), and extracellular acidification rate (ECAR) ( g ) were examined. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: RNA Sequencing, Stable Transfection, Transfection, shRNA, Control, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Binding Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Cellular extracts from A549 cells stably expressing FLAG (control) or FLAG-Notch1 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS-PAGE and silver stained. The differential protein bands were retrieved and analyzed by mass spectrometry. b A549 cells were transfected with Notch1 ICD, p300 shRNA, pCAF shRNA, or empty vector (EV). Glycolytic gene expression was measured using qRT-PCR. * P < 0.05. c ChIP analysis of Notch1, p300, and pCAF occupancy on glycolytic gene promoters in A549 cells. The graph shows the percentage of input. d Re-ChIP analysis of the occupancy of Notch1 and p300 or pCAF on the glycolytic gene promoters in A549 cells. e A549 cells were immunoprecipitated with anti-p300, anti-pCAF, or normal IgG, and the precipitates were analyzed by immunoblot with the indicated antibodies. IP immunoprecipitation. f A549 cells stably transfected with Notch1 were co-transfected p300 shRNA or pCAF shRNA. The protein expression of glycolytic genes was examined using western blot assay. g Notch1, p300, pCAF, and histone H3 and H4 acetylation occupancy on the promoters of indicated glycolytic genes in A549 cells transfected with Notch1 shRNA, p300 shRNA or pCAF shRNA was examined using ChIP assay.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Stable Transfection, Expressing, Control, SDS Page, Staining, Mass Spectrometry, Transfection, shRNA, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a The reporter activity of Notch1 and Hes1 in A549 cells transfected with TAZ was measured by luciferase reporter assay. * P < 0.05. Relative luciferase activity was performed to identify Notch responsive region in the Hes1 promoter. b The protein levels of Notch1 and Hes1 induced by TAZ overexpression in A549 cells were examined by western blot assay. c Notch1 ICD associated with endogenous TAZ in A549 cells. Immunoprecipitation (IP) was performed using antibodies of Notch1 ICD and TAZ, and coprecipitated protein was analyzed by western blot assay. d Comparison of Notch1 ChIP-seq signal (expressed as normalized read density, RPKM) in active enhancers with or without TAZ peaks in A549 cells treated with DMSO, Brontictuzumab (2 μm for 5 h), or cells transfected with TAZ shRNA. * P < 0.05. e TAZ binding at enhanc e rs of TAZ target genes in TAZ 4SA overexpressing A549 cells by ChIP-qPCR analysis. DNA enrichment was calculated and presented as fold vs control cells. f ChIP-qPCR analysis showed Notch1 binding on enhancers and promoters (TSS, transcription start site) of TAZ targets upon TAZ 4SA overexpressing in A549 cells, but not in the presence of Brontictuzumab (2 μm for 5 h).

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Over Expression, Western Blot, Immunoprecipitation, Comparison, ChIP-sequencing, shRNA, Binding Assay, ChIP-qPCR, Control

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Protein analysis of Jagged1, TAZ, TEAD1, and Hes1 in A549 cell lysates after transfected with TAZ and concomitant silencing of TEAD1 by shRNA. b Hes1 reporter assay in the presence (+) or absence (−) of Notch1 ICD, or TAZ, N = 3. Results of luciferase reporter assays are shown. * P < 0.05. c TEAD1-reporter luciferase assay in the presence (+) or absence (−) of TAZ or nTEAD1, N = 3. d Jagged1 luciferase reporter assay in the presence (+) or absence (−) of Notch1 ICD, TAZ, or Mst1, N = 3. * P < 0.05. Cells were transfected with or without MST1, phosphorylated TAZ and TAZ were analyzed by western blot assay. e ChIP assay for Notch1 or TAZ in A549 cells transfected with Notch1, TAZ, and plasmid of either Jagged1-ECR1 or ECR6. Data are presented as fold enrichment over an IgG ChIP performed with the same samples. f ChIP assay for TAZ in A549 cells at Jagged1-ECR1, ECR6, and Hes1 promoter. N = 3. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Transfection, shRNA, Reporter Assay, Luciferase, Western Blot, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a The proliferation curve of A549 cells transfected with Notch1, TAZ, TAZ shRNA or empty vector. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. b The proliferation curve of A549 cells transfected with Notch1 or empty vector, treated with 2.5 mM 2-DG as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. c The proliferation curve of A549 cells transfected with TAZ or empty vector, treated with 2.5 mM 2-DG as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. d The proliferation curve of A549 cells transfected with Notch1, TAZ or empty vector, treated with 0.1 mM Oligomycin in normal culture medium (containing 25 mM glucose) as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. e MicroPET-CT imaging of nude mice to determine FDG uptake in mice with subcutaneous xenograft lung cancer model established with A549 cells transfected with empty vector, p300 shRNA, pCAF shRNA, Notch1, TAZ, Notch1 shRNA, or TAZ shRNA. Representative 18F FDG microPET images are shown with arrowheads indicating xenografted lung cancers at treatment end (day 35). Quantification of 18F FDG uptake in tumors is shown as %IDmean/g. * P < 0.05. f Xenograft tumors were established as in ( b ) and the growth curve was plotted. * P < 0.05. g A549 cells stably expressing Notch1, TAZ, or LDHA shRNA were subcutaneously injected into nude mice. 2-DG was used as indicated. The growth curve was plotted. * P < 0.05. h Representative expression of Notch1 and TAZ by immunohistochemistry assay of 23 lung cancer patients. The correlation of glucose uptake with Notch1 or TAZ expression was determined using the Mann–Whitney U test. Scale bar = 50 μm. Original magnification: ×100. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Transfection, shRNA, Plasmid Preparation, CCK-8 Assay, Imaging, Stable Transfection, Expressing, Injection, Immunohistochemistry, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: Notch1 forms a positive feedback loop with TAZ and promotes glycolytic gene expressions through interaction with p300 and pCAF. Increased levels of extracellular lactate via Notch1/TAZ loop inhibits cytotoxic T-cell activity, which contributes to lung cancer invasion.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Activity Assay