Inhibition of transcription attenuates AMPK-induced UCP2 mRNA and UCP2 protein in HUVECs.

<p><b>A.</b> Effects of AICAR, metformin and berberine on UCP2 expression in HUVECs. n = 3; *p<0.05, vehicle <i>vs</i>. treatment. <b>B.</b> Quantitative PCR analysis of UCP2 mRNA levels in confluent HUVECs treated with AICAR (for 1 h), berberine (for 1 h) or Rosiglitazone (for 24 h). Total mRNA was isolated and UCP2 mRNA was quantified by real-time PCR compared with GAPDH. n = 4; *p<0.05, vehicle <i>vs</i>. treatment. <b>C.</b> Effect of actinomycin D on up-regulation of UCP2 by berberine. Confluent HUVECs were pretreated with actinomycin D (10 µg/ml) for 1 h, then treated with berberine or AICAR for 2 h. Cell lysates were subjected to Western blot analysis using antibody against UCP2 and β-actin. The blot is a representative of three blots from three independent experiments. <b>D.</b> Effect of cycloheximide on expression of UCP2 by berberine and AICAR. Confluent HUVECs were pretreated with cycloheximide (10 µmol/L) for 1 h, then treated with berberine or AICAR. Cell lysates were subjected to Western blot analysis using antibody against UCP2 and β-actin. (n = 3; *p<0.05 vehicle <i>vs</i>. berberine or AICAR treatment, #p<0.05 cycloheximide <i>vs</i>. vehicle). <b>E.</b> Immunoblot analysis of UCP2 expression in HUVECs transfected with NRF1 siRNA. HUVECs were transfected with control siRNA or NRF1 siRNA for 48 h, then treated with berberine for 2 h. Cell lysates were subjected to Western blot analysis using antibodies against NRF1 and UCP2. The blot is a representative of three blots from three independent experiments.