Fig. 5.

Effect of RAPA on 4-HNE-induced loss of neurites in SH-SY5Y cells. (a) experiment design. SH-SY5Y cells were plated in normal culture medium which was replaced the next day with differentiation medium which contained reduced serum (2.5% FBS) and 10 mM retinoic acid. Two days later, different doses of RAPA and corresponding volume of vehicle in normal culture medium was added to cells, followed by 4-HNE (15 µM) treatment 19 h later for another 5 h followed by protein extraction. Blank controls had no reagent, including vehicle, added to cells. (b) representative images captured using PerkinElmer High Content Screen Imaging system (20X). Orange staining is for cell membrane (neurite), blue staining is for nuclei. (c,d) effect of RAPA on 4-HNE-induced decrease in maximum neurite length (c) and total neurite length (d) in SH-SY5Y cells. Cells (n=1119±94.6, mean±SEM) in 3–4 images per experiment were analyzed using Columbus Image Data Storage and Analysis System. Data were analyzed by two-way ANOVA with followed by Tukey post hoc tests to compare individual means. ****p=0.0000, **p<0.01. Experiments were repeated three times.