Figure 1. Scheme of miR-155-PLBr and shPLBr expressing vectors as well as of a luciferase PLB cDNA reporter gene plasmid (A) and luciferase expression in HEK293 cells co-transfected with either PLB silencing or non-silencing control plasmids (B).

(A) Schematic representation of the amiR155-PLBr, shPLBr and luciferase reporter vectors. Upper panel: The amiR155-PLBr and shPLBr were inserted into an AAV shuttle plasmid containing an AAV2 vector backbone. The structure and nucleotide sequence of the amiR155-PLBr and shPLBr are shown below the vector scheme. The antisense strand of the mature amiR155-PLBr and shPLBr is highlighted in grey. Middle Panel: The shPLBr expression is driven by an U6 polymerase III promoter and terminated by five consecutive thymidines. The amiR155-PLBr expression is driven by a CMV-enhanced 0.26 kb rat MLC hybrid polymerase II promoter (CMV_enh-MLC0.26) and terminated by a SV40 poly A signal (pA). Note: The 3′ ITR of the AAV genome has a mutation in the terminal resolution site resulting in self complementary AAV vector genomes after packaging into AAV6 capsids. Lower panel: Schematic illustration of plasmids containing the Firefly luciferase reporter gene and the PLB cDNA (pLuc-PLBr_cDNA) in its 3′UTR. (B) Silencing activity of shPLBr and amiR155-PLBr using reporter assays. HEK293 cells were transfected with pLuc-PLBr_cDNA and Renilla luciferase expression plasmid and cotransfected with either amiR155-PLBr or shPLBr expressing plasmids. In addition, cotransfection was also done using the respective control plasmids amiR155-Con, shCon and amiR155-Luc. Cells were harvested 72 h after transfection and luciferase activity was determined. Firefly luciferase expression signals of the pLuc-PLB_cDNA reporter were normalized to Renilla luciferase and related to the respective signals in exclusively with Firefly luciferase expression vector transfected cells (Mock). *p<0.01 vs. respective control.