Fig. 3.

Induction of ER-stress dependent transcription factors by acrolein: (A) Induction of ATF4. Representative Western blots of lysates prepared from HUVECs harvested 4h after treatment with 10 or 25 μM acrolein or 1 μM thapsigargin (TG) as described under Materials and Methods developed using anti-ATF4 antibodies. The intensity of the ATF4 positive bands was normalized to actin as shown. (B) Induction of ATF3. Western blots of lysates of HUVECs treated for 2h with the indicated concentrations of acrolein or 1 μM thapsigargin (TG). Group data of normalized band intensity was shown in the lower panel. (C) Nuclear translocation of ATF3. Nuclear extracts were separated from HUVECs treated with 0 or 10 μM acrolein and harvested after the indicated time. The intensity of the bands obtained upon Western analysis using anti-ATF3 antibody was normalized to the nuclear protein HDAC1. (D) Induction of CHOP by acrolein. Cells were harvested 6h after treatment with 10 μM acrolein as described under Materials and Methods and the CHOP mRNA was quantified by RT-PCR using CHOP-specific primer. Representative Western blot (lower panel) of lysates from HUVEC cells harvested 6h or 12h after treatment with 0 or 10 μM Acrolein developed using an antibody against CHOP using thapsigargin (TG) treated cells (6h, 1 μM) as positive control. (E) Inhibition of acrolein-stimulated ATF3 induction by PBA. HUVECs were left untreated or treated with 10 mM PBA as described under Materials and Methods and then incubated with 10 μM acrolein. Two hours after acrolein treatment, the cells were harvested and the abundance of ATF3 protein was examined by Western analysis. The band intensity due to ATF3 was normalized to actin. Data are presented as mean ± SE. * p<0.05, ** p<0.01, ***p<0.001 compared with control, and # p<0.05 PBA treated versus untreated cells (n=3–4).