RNA binding assay to measure the capacity of JTR-009 to replace IRP1 binding to biotinylated probes encoding core APP IRE sequences compared to IRP1 binding to the H-ferritin IRE sequences (N = 4).
Panel A: Top Panel: Cartoon representation of the protocol employed to detect RNA binding between IRE probes and IRP1 in SH-SY5Y cell lysates. Bottom Panel: Effect of JTR-009 treatment of SH-SY5Y cells (24 h, 10 µM) to alter ferritin-H IRE binding to IRP1 compared to that of the APP IRE. Panel B: The calcein assay for iron levels in SH-SY5Y cells in response to treatment with JTR-009. Cells were treated with either DMSO (negative control), extracellular iron chelator (DPTA), or JTR-009 at 10 µM for 48 hours. Panel C: The anti-amyloid-Aβ-42 efficacy of the APP 5′UTR inhibitors JTR-005 and JTR-009, as measured in conditioned medium from SH-SY5Y neuronal cells (Chemiluminescent BetaMark x-42 ELISA assay from Covance, inc). Data shows the mean values for the reduction of levels of Aβ-42 ± SEM (N = 3) after 72 h treatment of the cells with JTR-009 compared to JTR-005 at 10 µM (p<0.01), analyzed by ANOVA.