Figure 1

M1 induces expression of ER chaperone genes.

(A) 293T cells were transfected with the GRP78-fluc or GRP78mut-fluc reporter plamids, the PGK-renilla-luciferase as an internal control and increasing amounts of plasmids encoding M1 or M3. The cell lysates were prepared 24 hours posttransfection for dual-luciferase assay. The ratio of firefly luciferase activity to renilla luciferase activity was calculated based on the value of the vector control (set as 1). This assay and all following assays were performed in triplicates. Error bars show standard deviation. (B) Cells were transiently transfected with the M1 or M3 expression plasmids for 24 hours and were harvested for RNA extraction. Quantitative RT-PCR was performed using a primer set specific for GRP78 (Table 1). The levels of GRP78 mRNA were normalized to that of GAPDH mRNA. The fold changes shown were calculated relative to the values obtained in vector-transfected cells (set as 1). (C) Cells were transfected as described in Fig. 1B and were harvested for western blot analysis using antibodies specific for GRP78 and β-actin (loading control) as indicated. M1 and M3 genes were tagged with Human influenza hemagglutinin (HA) and the expressions were shown using anti-HA antibodies. The lower panel shows the intensity of bands that was quantified from western blot analysis by measuring the peak height of the bands using ImageJ software. The intensity of bands in top and middle panel was compared to the intensity of vector control (which was defined as 100%). The relative intensity was calculated by normalizing the intensity of upper panel bands (GRP78) to the middle panel bands (β-actin). (D) The promoter activities of GRP94 and ERdj4 were determined by reporter assay as described in Fig. 1A using the GRP78-fluc and ERdj4-fluc reporter plasmids and mRNA levels determined quantitative RT-PCR analysis as in Fig. 1B using specific primer sets for indicated genes (Table 1). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.