Research Paper Volume 12, Issue 7 pp 6385—6400

Figure 3. NORAD-knockdown aggravates ox-LDL-induced oxidative stress and p-IKBα expression level in HUVECs. HUVECs were transfected with siNORAD or scrambled siCTRL. After transfection for 24 h, the cells were treated with 60 μg/mL ox-LDL for 24 h. (A) NORAD knockdown aggravated ox-LDL-induced ROS production. HUVECs were treated with 60 μg/mL ox-LDL for 3 h and incubated with DCF-DA for 25 min. The ROS levels were observed under an inverted fluorescence microscope. Images were representative of three separate experiments (n = 3). (B) Flow cytometry was used to detect the intracellular ROS levels. Data were from three separate experiments and described as mean ± SD. ^*P < 0.05 vs. the siCTRL group. (C) MDA content in HUVECs treated with 60 μg/mL ox-LDL for 24 h. Data were shown as mean ± SD of three separate experiments. ^**P < 0.01 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL. (D) NORAD-knockdown increased the p-IKBα expression observed through western blot. The ratio of p-IKBα to IKBα was analyzed with ImageJ. Values were shown as mean ± SD (n = 3). ^**P < 0.01 vs. siCTRL. (E) NORAD-knockdown increased NF-κB nuclear translocation by immunofluorescence. p65 was stained red with Cy3. Nuclei were stained blue with DAPI. Co-localization of p65 with nucleus is shown in purple.