Figure 6
From: Degradation of insulin amyloid by antibiotic minocycline and formation of toxic intermediates
Degradation of insulin amyloids formed by insulin preparations (lispro and detemir) by minocycline. (A) BTD21 assay of lispro (upper panel) and detemir (lower panel) incubated in the presence of 0–100 μM minocycline. The left figures are the spectrum of BTD21 of insulin amyloid incubated for 72 h: amyloid (thick solid line), 25, 50, and 100 μM minocycline (thick-dashed, thick dotted line, solid-dashed lines, respectively), and BTD21 probe (thin-solid line). The right figures are the plots of the intensities normalized for insulin amyloid as 100%: amyloid (white squares) and 25, 50, and 100 μM minocycline (black, gray, white circles, respectively). (B) BCA assay of supernatant samples to confirm the dependency of the degradation on minocycline concentration (upper) and incubation time (lower): lispro (gray) and detemir (white). Protein concentrations were normalized for 50 µM (all insulin amyloid concentrations). (C) Cytotoxicity of degraded samples using MTT assay against HeLa cells. Lispro (upper left) amyloid and detemir (upper right) were incubated with minocycline for 1 week at the indicated concentrations. Lispro (lower left) and detemir (lower right) were incubated with a fixed minocycline concentration (50 µM) for the indicated periods. 0.1, 0.5, and 1 μM are indicated by gray, white, and black, respectively. The absorbance was normalized for PBS as 100%. (*P < 0.05, **P < 0.01, ***P < 0.005).
