The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein (SNCA).
Panels A and B: Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and SNCA levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). Right Panel: Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. Panel C: Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p<0.01), 0.1 µM (** = p<0.015), and 1 µM (*** = p<0.01) analyzed by ANOVA (N = 5). Dotted line: Representative LDH assay parallel to Aβ determination for SH-SY5Y cells treated for 72 h at concentrations up to 100 µM of JTR-009 (N = 4). Panel D: MTS assay for cellular mitochondrial viability after treatment of SH-SY5Y cells with JTR-005 and JTR-009 at the concentrations shown. Y axis: Percent of maximal viability ± SEM after treatment of the cells with JTR-009 and JTR-005 (N = 3)). Shown are the relative trend-lines for the dose-responsive viability of JTR-005 and JTR-009 compared to untreated cells (‘poly’ = non linear polynomial regression of the data).