luciferase reporter vector pgl2-basic  (Promega)

Journal: Neoplasia (New York, N.Y.)

Article Title: pH, Lactate, and Hypoxia: Reciprocity in Regulating High-Affinity Monocarboxylate Transporter Expression in Glioblastoma 1

doi: 10.1016/j.neo.2016.12.011

Figure Lengend Snippet: Construction of nested deletion mutants of MCT2 promoter-luciferase reporter gene constructs: (A) The 4.2-kbp promoter-exon I fragment was cloned into pGL2-firefly luciferase reporter gene vector at indicated restriction sites, followed by digestion with exonuclease III to generate a series of 5′ nested deletion mutants to identify the promoter regions most responsive to pH, hypoxia, glucose, and lactate. (B) Agarose gel electrophoresis pattern of sequentially generated nested deletion mutants (λ-HinD III markers were used as reference standards). Putative transcription factor binding sites on the 4.2-kbp promoter region identified by the MatInspector program (Genomatix Software Suite) are listed above the nested deletion mutant map.

Article Snippet: The promoter-exon I region was cloned into luciferase reporter gene vector pGL2-Basic (#E1641; Promega, Madison, WI) in frame with the luciferase coding sequence using a series of cloning steps (Suppl.

Techniques: Luciferase, Construct, Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Generated, Binding Assay, Software, Mutagenesis

Journal: Cell Research

Article Title: Hepatocellular carcinoma redirects to ketolysis for progression under nutrition deprivation stress

doi: 10.1038/cr.2016.109

Figure Lengend Snippet: Activation of mTORC2-AKT-SP1 signaling induces OXCT1 expression and ketolysis in nutrition-limiting HCC cells. (A) Western blot analysis of p-AMPK (threonine 172 phosphorylation of AMPK), AMPK, p-AKT (serine 473 phosphorylation of AKT), AKT, p-ERK1/2 (threonine 202/tyrosine 204 phosphorylation of ERK1/2), ERK1/2, IκBα and NF-κB levels in HepG2 cells under normal (0 h) or serum starvation conditions for 6, 12 and 24 h. (B) qRT-PCR and western blot analysis of OXCT1 expression in HepG2 cells incubated with vehicle (DMSO), compound C (10 μM), LY-294002 (50 μM), PD98059 (50 μM), EVP4593 (1 μM) or Z-VAD-FMK (50 μM) for 2 h prior to SS for 24 or 48 h. Data were presented as mean ± SD. *P< 0.05 as compared with vehicle-treated normal group; #P< 0.05 as compared with vehicle-treated SS group. (C) AKT and OXCT1 protein levels were determined by western blot in HepG2 cells stably expressing AKT shRNAs (sh1 and sh2) or NTC under normal or SS conditions for 48 h. (D) Western blot analysis of OXCT1 expression in HepG2 cells treated with vehicle (DMSO) or 50 nM rapamycin for 24 and 48 h in the presence or absence of serum. (E) HepG2 cells stably expressing Rictor shRNAs or NTC were cultured under Nor or SS conditions for 48 h. Rictor and OXCT1 expression were determined by western blot. (F) Western blot analysis of SP1 and OXCT1 expression in HepG2 cells expressing SP1 shRNA or NTC, or in HepG2 cells pretreated with vehicle (DMSO) or MIT (20 nM) for 2 h, under normal or SS conditions for 48 h. (G) Western blot analysis of SP1 and OXCT1 levels in HepG2 cells expressing pSIN-SP1 or pSIN-EV. (H) HEK293 cells were co-transfected with SP1 shRNAs and pGL2-OXCT1-promoter or pGL2-EV luciferase reporter vectors, and 24 h after transfection, medium was changed to normal (Nor) or SS for another 24 h, followed by dual luciferase assay. Data were presented as mean ± SD. *P< 0.05 as compared between the indicated groups. (I) HEK293 cells were co-transfected with pSIN-SP1 and pGL2-P-GCbox (GCbox-1/2 wt), pGL2-P-GCbox mutants (GCbox-1/2 mt) or pGL2-P-EV luciferase vectors. Dual luciferase assay were performed 24 h after transfection. Data were presented as mean ± SD. *P< 0.05 as compared between the indicated groups. (J) The occupancy of potential GC-box (GCBox-1/2) by SP1 was analyzed by ChIP assay in HepG2 cells cultured under normal (Nor) or SS conditions for the indicated hours using anti-SP1 antibody or IgG control. Data were presented as mean ± SD. *P< 0.05 as compared with corresponding Nor group. (K) HepG2 cells stably expressing SP1 shRNAs or NTC were further infected with viruses expressing pBaBe-myr-flag-AKT (myr-AKT) or pBaBe-EV, followed by western blot analysis of AKT1, p-SP1 (threonine 453 phosphorylation of SP1), SP1 and OXCT1 levels. (L) Schematic representation for serum starvation-activated OXCT1 transcription mediated by mTORC2, AKT and SP1. (M) HepG2 cells stably expressing AKT shRNA, SP1 shRNA or NTC were cultured under Nor or SS conditions for 48 h followed by incubating with 5 mM of [2, 4-13C2] β-HB for 24 h and subsequent isotope tracing of 13C-labeled metabolites by GC-MS. Data were presented as mean ± SD. *P< 0.05 as compared with Nor-treated NTC group; #P< 0.05 as compared with SS-treated NTC group.

Article Snippet: Human OXCT1 promoter sequences were inserted into the pGL2 basic luciferase reporter vector (Promega), designated as pGL2-OXCT1 promoter.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Incubation, Stable Transfection, Cell Culture, shRNA, Transfection, Luciferase, Infection, Labeling, Gas Chromatography-Mass Spectrometry