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. 2020 Jun 18;11(6):469. doi: 10.1038/s41419-020-2663-1

Fig. 4. Effect of NaB on high cholesterol-induced NOX2 expression.

Fig. 4

a SK-N-MC cells were pretreated with Vas2870 (5 µM) or mitotempo (2 µM) for 30 min prior to high cholesterol treatment for 72 h where ROS with DCF-DA were measured by flowcytometer. Total cell counts = 1.0 × 104 cells. n = 4. *p < 0.05 versus control, #p < 0.05 versus high cholesterol treatment. b Cells were pretreated with NaB for 30 min prior to high cholesterol treatment for 72 h where mtROS with mitoSoxTM red were measured by flowcytometer. Total cell counts = 1.0 × 104 cells. n = 4. #p < 0.05 versus high cholesterol treatment. c Cells were pretreated with NaB for 30 min prior to high cholesterol treatment for 24 h. NF-κB, Lamin A/C, and β-actin protein levels in cytosolic and nuclear-fractionized samples were analyzed by western blot. n = 3. *p < 0.05 versus control, #p < 0.05 versus high cholesterol treatment. d Cells were pretreated with NaB for 30 min prior to high cholesterol treatment for 24 h and immunostained with NF-κB antibody. Scale bars are 8 µm (magnification, ×1,000). n = 3. *p < 0.05 versus control, #p < 0.05 versus high cholesterol treatment. e Cells were pretreated with NaB for 30 min prior to high cholesterol treatment for 24 h. Data were normalized by the ACTB mRNA expression levels. n = 4. f Cells were pretreated with NaB for 30 min prior to high cholesterol treatment for 24 h. The expression levels of NOX2 were analyzed by western blot. β-actin was used as a loading control. n = 4. *p < 0.05 versus control, #p < 0.05 versus high cholesterol treatment. g Brains of 20 weeks mice fed with HFD for 14 weeks were extracted. The expression levels of NOX2 were analyzed by western blot. β-actin was used as a loading control. n = 3. *p < 0.05 versus control. h, i Cells were pretreated with Bay11-7082 (5 µM) for 30 min prior to high-cholesterol treatment for 24 h. The expression levels of NOX2 and BACE1 were analyzed by western blot. β-actin was used as a loading control. n = 4. j Cells were pretreated with Bay11-7082 for 30 min prior to high cholesterol treatment for 72 h. Aβ concentration of medium samples was detected by using ELISA kit. n = 4. k Cells were pretreated with Vas2870 or NAC (5 mM) or mitotempo for 30 min prior to high-cholesterol treatment for 24 h. The expression levels of BACE1 were analyzed by western blot. β-actin was used as a loading control. n = 4. l Cells were pretreated with Vas2870 or NAC or mitotempo for 30 min prior to high-cholesterol treatment for 72 h. Aβ concentration of medium samples was detected by using ELISA kit. Data are presented as a mean ± S.E.M. n = 4. *p < 0.05 versus control, #p < 0.05 versus high cholesterol treatment. All blot and immunofluorescence images shown are representative.