Figure 1

Degradation of two types of insulin amyloids by minocycline. (A) LCO assay and MTT assay of (i)-amyloid and (r)-amyloid. The left image is the spectrum of pFTAA, and the middle image is the spectrum of BTD21: (i)-amyloid (thick solid line), (r)-amyloid (thick dotted line), and probe (thin solid line). The right images show results of the MTT assay of (i)-amyloid, (r)-amyloid, and native insulin. All samples were quantified by BCA assay and were diluted to the same protein concentration (1 μM/well). The absorbance was normalized for PBS as 100%. (B) ThT assay of (i)-amyloid (upper) and (r)-amyloid (lower) incubated in the presence of 0–100 μM minocycline. Images on the left are the spectrum of ThT incubated with insulin amyloid for 72 h: amyloid only (thick solid line), 25, 50, and 100 μM minocycline (thick-dashed, thick dotted, solid-dashed lines, respectively), and probe (thin solid line). Pictures on the right are the plots of the intensities normalized for insulin amyloid as 100%: amyloid (white squares), and 25, 50, 100 μM minocycline (black, gray, and white circles, respectively). (C) BCA assay of supernatant samples to confirm the dependency of the degradation on minocycline concentration (upper) and incubation time (lower): (i)-amyloid (gray) and (r)-amyloid (white). Protein concentrations were normalized to 50 µM (all insulin amyloid concentrations). (D) TEM images of the structure of insulin amyloid after degradation. The three samples on the left area are (i)-amyloid, and the three samples on the right area are (r)-amyloid. The mature amyloid (left) was incubated with 2.5 (middle) or 25 µM (right) minocycline for 1 week. The scale bar is 200 nm.