Pgl4.74 hrluc tk plasmid

Viral RNA polymerase activity was tested in HEK293T cells using the reconstituted minigenome assay with the pPolI-WSN-NA-firefly-luciferase reporter construct (gift from Dr. Yoshihiro Kawaoka, University of Wisconsin-Madison, Madison, WI, USA) and in mouse L-cells using the pHL-NS-FF-Luc reporter construct (gift from Dr. Georg Kochs, University of Freiburg, Freiburg, Germany). The assay was performed as described in [41 (link)], except the pHW-C71, pHW-C72, pHW-C73 and pHW-C75 plasmids were used for the expression of CA/07 PB2, PB1, PA and NP proteins, respectively, and the pGL4.74(hRluc/TK) plasmid (Promega, Madison, WI, USA) for control Renilla luciferase expression. The dual luciferase assay was performed 24 h post-transfection using the Dual-Glo Luciferase Assay System (Promega). Site-directed mutagenesis was utilised to introduce amino acid substitutions in PB1 and PA expression vectors pHW-C72 and pHW-C73 as described in Section 2.4 above to test their contribution to reconstituted viral polymerase activity.

The activity of the Wnt signaling pathway was detected by a Wnt signal reporter assay using the TOP/FOP TCF reporter kit (Millipore, USA). Cells were seeded in 24-well tissue culture plates at 5 × 10⁴ cells/well in 0.5 mL of Opti-MEM (Invitrogen) at 12 h before transfection. On the day of transfection (day 1), 900 ng of the TOP flash or FOP flash construct (Millipore) together with 100 ng of the pGL4.74[hRluc/TK] plasmid (Promega) was used to transfect the cells in each well. The pGL4.74[hRluc/TK] plasmid containing the Renilla luciferase gene was used as an internal control for normalizing the transfections. Transient transfections using Lipofectamine LTX and PLUS reagent (Invitrogen) were performed according to the manufacturer’s instructions. Eight hours after transfection, the transfection medium was changed to DMEM/F-12 containing 5% FBS, and the cells were exposed to HBO. On days 3 and 5, the cells were re-transfected once and exposed to HBO as described above. After an additional 24 h of culturing, the cells were washed with PBS and harvested using 100 μL/well of passive lysis buffer (Promega). The cell lysates (20 μL) were evaluated for luciferase activity using a Dual-Luciferase Reporter Assay Kit (Promega). Luciferase activity was measured according to the manufacturer’s instructions.