Fig. 3

Combined overexpression of ST3GAL1 and TMPRSS2 and the resulting viral titer in cells. a Schematic representation of the piggyBac transposon based expression vector harboring ST3GAL1-T2A-TMPRSS2. The vector was used to express both ST3GAL1 and TMPRSS2 in WT DF-1 cells, termed O/E-ST3T2. b (top) Expression of ST3GAL1 and TMPRSS2 in O/E-ST3T2 and WT DF-1 cells was analyzed by qRT-PCR. Data were normalized to expression of chicken ACTB and expressed as the mean ± standard deviation (n = 3). Significant differences (compared with WT DF-1 cells) were determined using Student’s t-test (**P < 0.01 and *P < 0.05). (bottom). Expression of ST3GAL1 and TMPRSS2 in O/E-ST3T2 and in WT DF-1 cells, as measured by RT-PCR. The chicken ACTB was used as a reference gene. The full length (uncut) gel electrophoresis image is shown in Additional file 3: Fig. S3. c Titer of PR8-H5N8 (PB2-627E) or PR8-H9N2 (PB2-627E) in O/E-ST3T2 cells and WT DF-1 cells in the absence (WT DF-1(−)) and presence (WT DF-1(+)) of trypsin. d Cell proliferation at 24 h, 48 h, and 72 h post-infection. Error bars indicate the mean ± standard deviation of triplicate analyses. e Viral titer at 24 h, 48 h and 72 h. Significant differences (compared with WT DF-1 cells) were determined by two-way ANOVA. A P value < 0.05 was considered significant (****P < 0.0001 and ***P < 0.001). Error bars indicate the mean ± standard deviation of triplicate analyses