Journal: Scientific Reports
Article Title: Arthropod transcriptional activator protein-1 (AP-1) aids tick-rickettsial pathogen survival in the cold
Figure Lengend Snippet: Approximately 700 bp DNA sequence upstream of tick antifreeze gene is sufficient to drive iafgp gene expression. ( A ) Schematic representation of the iafgp promoter construct used to analyze promoter activity in tick cells. The schematics is not drawn to scale. ~700 bp DNA sequence (position corresponding to 17757–18463 in GenBank acc. no. DS704943) upstream of iafgp gene containing all tested binding sites (AP-1, HSF-1, TATA-region) was cloned into promoterless pGLuc (pGLuc-P iafgp ) vector. The empty pGLuc vector was used as control. These constructs were transfected into tick cells and luciferase assay was performed. ( B ) Luciferase activity measurements determined from culture supernatants from uninnfected (UI) or A . phagocytophilum -infected (I) ISE6 cells transfected with pGLuc-P iafgp or pGLuc constructs is shown. ( C ) Levels of luciferase transcripts in uninfected (UI) or A . phagocytophilum -infected (I) ISE6 cells transfected with pGLuc-P iafgp or pGLuc constructs is shown. The levels of luciferase transcripts were normalized to the levels of beta-actin transcripts. Open or dark grey circles represents uninfected (UI) or A . phagocytophilum -infected (I) cells transfected with pGluc alone, respectively. Light grey or black circles represents uninfected (UI) or A . phagocytophilum -infected (I) cells transfected with pGLuc-P iafgp , respectively. ( D ) Luciferase activity measurements determined from culture supernatants from A . phagocytophilum -infected ISE6 cells treated with mock-dsRNA or ap-1- dsRNA and transfected with pGLuc-P iafgp constructs is shown. In panels B and D, each circle represents luciferase activity measurement from supernatants collected from one culture well. In panel C, each circle indicates sample generated from cells from one culture well. Statistical analysis was performed using Student’s t test and P value less than 0.05 was considered significant in panels B–D.
Article Snippet: After incubation, 750 ng of ap-1 dsRNA or equal volume of mock solution or control dsRNA or pGLuc/pGLuc-Piafgp constructs were mixed with Lipofectamine 2000 (Thermo Fisher Scientific, USA) reagent and added to the wells.
Techniques: Sequencing, Expressing, Construct, Activity Assay, Binding Assay, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Infection, Generated