(A) Human IAPP (hIAPP) aged for 24 h contains amyloid fibrils and no detectable oligomers. Amyloid fibrils were observed using ThT binding and TEM. Oligomers were detected using a dot bot assay with the polyclonal anti-oligomer antibody, LOC. hIAPP oligomers were used as the positive control for LOC binding. The dashed line on the ThT binding graph indicates ThT fluorescence of vehicle alone. (B) and (C) hIAPP aged for 24 h is significantly more cytotoxic than hIAPP aged for 0 h. In these experiments, 50 μM human and mouse IAPP were aged for the designated time periods and then they were applied to cells at 5 μM final concentration. Mouse IAPP (mIAPP), which does not form amyloid fibrils, is not cytotoxic regardless of time period of aging. Black horizontal bars indicate the median (n = 12–15 across 4–5 biological replicates, each with three technical replicates). (B) Rin5F cells treated with hIAPP aged for 24 h reduce significantly less MTT dye than Rin5F cells treated with hIAPP aged for 0 h (ns=not significant; ****p<0.0001 using an unpaired t-test with equal standard deviations). (C) Rin5F cells treated with hIAPP aged for 24 h exhibit significantly higher caspase-3/7 activation than Rin5F cells treated with hIAPP aged for 0 h. Additionally, Rin5F cells treated with hIAPP aged for 24 h exhibit significantly higher caspase-3/7 activation than vehicle-treated cells (***p=0.0008 using an ordinary one-way ANOVA), but Rin5F cells treated with hIAPP aged for 0 h do not (p=0.4286 using an ordinary one-way ANOVA) (ns=not significant; **p=0.0011 using an unpaired t-test with equal standard deviations). (D) The insoluble fraction of hIAPP aged 24 h, which contains amyloid fibrils and no detectable oligomers, contains the cytotoxic species. Cytotoxicity was measured using MTT dye reduction and detection of caspase-3/7 activation (****p<0.0001; **p<0.0013; n = 9 across three biological replicates, each with three technical replicates). (E) Amino acid sequences of human IAPP and mouse IAPP. The location of the early onset familial mutation, S20G, is shown below the human sequence. Red residues in the mouse sequence differ from the human sequence. The amyloid spine of human IAPP and the corresponding region in the mouse sequence is enclosed in the gray box. (F). Schematic of protein segments that span the amyloid spine, hereon referred to as spine segments, targeted for characterization. (G) Fibrils of spine segments seed hIAPP fibril formation, suggesting that spine segments embody structural characteristics of full-length hIAPP fibrils. 10 μM hIAPP was seeded with 10% (v/v) monomer equivalent of pre-formed, unsonicated seed of each spine segment. mIAPP, which does not contain amyloid fibrils, does not seed hIAPP fibril formation. Curves show average of 4 technical replicates.