Journal: The Journal of Experimental Biology

Article Title: Circadian coupling of mitochondria in a deep-diving mammal

doi: 10.1242/jeb.246990

Figure Lengend Snippet: Clock genes expression profile in synchronized hooded seals fibroblasts and tissues. (A) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycle (°C), as represented in the upper panel. Arntl and per2 mRNAs show a 24-h period and are in antiphase. P -values and phases were calculated through JTK cycle analysis ( n =4). (B) mRNA expression of core clock genes arntl (blue line) and per2 (black line) in hooded seal fibroblasts synchronized by temperature cycling and then left at constant temperature (upper panel). Arntl and per2 mRNAs maintain a 24-h period and their antiphasic relationship also in constant conditions. P -values and phases were calculated through JTK cycle analysis ( n =4). (C) Photon multiplier tube recordings of hooded seal skin fibroblasts transfected with arntl :luciferase (blue line) and per2 :luciferase (black line). Cells were synchronized with dexamethasone. (D) Arntl mRNA expression in hooded seal kidney and liver tissue, sampled in mid-light phase (ZT6) and mid-dark phase (ZT18). * P <0.05 (one-way ANOVA; n =3 except liver ZT18, where n =2). Data are expressed as means±s.e.m.

Article Snippet: The latter consisted of the pLV6 backbone and a mouse per2 promoter with adjacent luciferase sequence contained in the pGL3 basic E2 vector (Addgene plasmid 48747).

Techniques: Expressing, Transfection, Luciferase

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