Journal: Cell Death and Differentiation

Article Title: PAK5 promotes the migration and invasion of cervical cancer cells by phosphorylating SATB1

doi: 10.1038/s41418-018-0178-4

Figure Lengend Snippet: PAK5-mediated phosphorylation promotes EMT in cervical cancer cells. a, b Effects of SATB1 and SATB1 Ser47A on the migration and invasion ability in HeLa and SiHa cells. Scale bar, 100μm for a and b. c HeLa and SiHa cells were transfected with pGL3-E, together with the indicated expression plasmids for Dual luciferase reporter assays. d Effects of SATB1 knockdown on EMT markers were analyzed by western blotting in HeLa cells overexpressing PAK5. e Effects of SATB1 overexpression on EMT markers in HeLa cells silencing PAK5. Data are showed as mean ± SD for three independent experiments. **, P < 0.01; ***, P < 0.001. pGL3-E pGL3-E-cadherin promoter, siCtrl siRNA control, siPAK5 siRNA PAK5, ns not significant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Briefly, E-cadherin promoter was subcloned into pGL3-Basic luciferase expression vector (Genepharma).

Techniques: Migration, Transfection, Expressing, Luciferase, Western Blot, Over Expression

Journal: Respiratory Research

Article Title: Knockdown of HSP110 attenuates hypoxia-induced pulmonary hypertension in mice through suppression of YAP/TAZ-TEAD4 pathway

doi: 10.1186/s12931-022-02124-4

Figure Lengend Snippet: TEAD4 promotes HSP110 transcription by binding to HSP110 promoter. a Schematic representation of the pGL3-basic-HSP110 promoter reporter constructs used in this study. The HSP110 promoter (− 2000/ + 20) and sequential deletion of the HSP110 promoter shorter fragments (− 1000/ + 20, − 500/ + 20) were cloned into the pGL3-basic luciferase vector. b Constructs with the HSP110 promoter fragments were co-transfected with overexpression vector containing TEAD4 or empty vector into HEK293 cells. The luciferase activity was analyzed. Data are means ± SD from 3 biological replicates. *p < 0.05 compared to vector, # p < 0.05 compared to HSP110 promoter fragments − 2000/ + 20 or − 1000/ + 20 under TEAD4 overexpression. c The chromatin immunoprecipitation of the HSP110 promoter with anti-TEAD4 or anti-IgG antibodies was performed using HPASMCs after hypoxic exposure. The sequences containing the TEAD4-binding sites on HSP110 promoter were amplified by PCR and detected by agarose gel. d The nuclear extract was prepared from HPASMCs after hypoxic exposure and biotinylated-HSP110 DNA probe specific to TEAD4-binding site 3 was used for pull-down assay. The protein level of TEAD4 was detected

Article Snippet: The HEK293T cells were seeded in 12-well plate and transiently transfected with PGL3-basic luciferase reporter vector harboring different human HSP110 promoter region fragments (− 2000/ + 20), (− 1000/ + 20) and (− 500/ + 20) with or without pcDNA3.1-TEAD4 plasmid (GenScript, China) using lipofectamine 3000 transfection reagent (Invitrogen) for 48 h. Empty vector pcDNA3.1 was used as a control.

Techniques: Binding Assay, Construct, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Over Expression, Activity Assay, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Pull Down Assay

Journal: Experimental and Therapeutic Medicine

Article Title: HBxAg promotes HBV replication and EGFR activation in human placental trophoblasts

doi: 10.3892/etm.2021.10645

Figure Lengend Snippet: (A) Dual-luciferase reporter gene assay of HBx activity on the EGFR promoter. HBx + E, co-transfection of pGFP-HBx and pGL3-EGFR promoter plasmids; control, co-transfection of pGFP empty vector and pGL3-EGFR promoter plasmids; NC, co-transfection of pGFP-HBx and pGL3-basic empty vector. Rate, firefly luciferase and internal reference Renilla luciferase ratio. ** P<0.01 vs. control. (B) Western blot analysis of EGFR/PI3K/p-AKT expression. Protein fold change is displayed as the ratio of the EGFR/PI3K/p-Akt/Akt protein band to the β-actin protein band; p-AKT/AKT, the ratio of p-AKT protein band to the AKT protein band. * P<0.05, ** P<0.01 vs. NC. EGFR, EGFR overexpressing cells; NC, negative control; sh, short hairpin; HBx, hepatitis B virus X; p-, phosphorylated.

Article Snippet: Cells were seeded into 24-well plates at 3x10 5 cells/well and incubated for 16-18 h. pGL3-EGFR promoter luciferase expression vector (BK328 pGL3-basic-EGFR; Umibio (Shanghai) Co. Ltd.) and the internal reference vector pRL-TK (Promega Corporation) were co-transfected with X-tremeGENE HP (Roche Diagnostics) at room temperature for 20 min when cell densities reached 50-60% confluence, at a ratio of 30:1 (expression vector 0.3 µg/well and internal reference 0.01 µg/well). pGL3-EGFR promoter luciferase expression vector and pGFP-HBx (Addgene, Inc.) were co-transfected as the experimental group, pGL3-EGFR promoter luciferase expression vector and pGFP empty vector (Addgene, Inc.) were co-transfected as the control group, pGL3-Basic and pGFP-HBx were co-transfected as a negative control (NC) and pGL3-Control was used as a positive control (Promega Corporation).

Techniques: Luciferase, Reporter Gene Assay, Activity Assay, Cotransfection, Plasmid Preparation, Western Blot, Expressing, Negative Control