Surface tension changes for various forms of insulin showing the extent of surface tension of solutions containing insulin with native, oligomer intermediates, proto-fibril, and fibril structures at the air-water interface.

<p>Data represent the average of decuple measurements and error bars represent standard deviation (SD). *Significantly different from native insulin. Statistical significances were achieved when p<0.05.</p

The effects of oligomer, proto-fibril and fibril forms of insulin on neurite complexity in differentiated PC12 cells.

<p>The criteria were quantified 12 h after treatment; <b>A</b>) primary neurites per cell; <b>B</b>) percent of bipolar cells and <b>C</b>) the ratio of nodes to primary neurites. * Significantly different from control cells. Statistical significances were achieved when p<0.05 (*p<0.05 and **p<0.01).</p

Representative far-UV CD spectra of β-Lg samples after incubation at pH 2 and 80°C.

<p>(A) In the absence and presence of Pb²⁺, curcumin and their mixture. (B) In the absence and presence of AFM1, curcumin and their mixture.</p

The effects of different form of β-Lg fibrils on neurite outgrowth in differentiated PC12 cells.

<p>(A) Average neurite length and (B) average neurite width (n = 3; *significantly different from control).</p

The effects of oligomer, proto-fibril and fibril forms of insulin on neurite outgrowth in differentiated PC12 cells.

<p><b>A</b>) The criteria of PC12 differentiation are shown on three neurons (left image) of a sample image. The “P” on right image indicates the primary neuritis of a neuron. The yellow arrow shows the length of a neurite, extent elongated, and membrane-enclosed protrusions of cytoplasm. The blue circle on right image shows the cell body. Neurite width is not equal in all parts of neurons, thus the average neurite width must be calculated by dividing cell body area to average neurite length. The white arrows show to bipolar cells. The letter “N” indicates the nodes, the sites at which individual neurites branched or separate neurites contacted each other. The criteria were quantified 12 h after treatment; <b>B</b>) NGF-differentiated PC12 cells were pretreated with three amyloid intermediate forms of insulin. C) Cell body area; D) average neurite length; and E) average neurite width. *Significantly different from control cells. Statistical significances were achieved when p<0.05 (*p<0.05 and **p<0.01).</p

The effects of different forms of β-Lg fibrils on the ratio of nodes to primary neurites of differentiated PC12 cells.

<p>The criteria were quantified after incubation for 4 or 8 h. (n = 3; *significantly different from control).</p

The kinetics of amyloid fibrillation induced by dissolving insulin (0.6 mg/mL) in glycine buffer (20 mM, pH 2.0) and 37°C and agitation.

<p><b>A</b>) The content of fibril formation was recorded via ThT fluorescence as a function of incubation time. Data represent the average of 3 independent measurements and error bars represent standard deviation from the mean value. <b>B</b>) The content of fibril formation was also recorded via Congo red absorption spectrum during incubation time. Congo red spectrum alone (<b>^____</b>) with native insulin (<b>….</b>) and with incubated samples (<b>- - -</b>) after 10, 13, 15, 18, 20, 23, 25 and 30 h, respectively. <b>C</b>) Far-UV CD spectra of bovine insulin during incubation time. At the onset of incubation (0h) two minima at 208 and 222 nm indicate α–helical structure. After 28 h of incubation, appearance of a new minimum at 216 nm indicates cross β–sheet structure because of amyloid fibrillation.</p

AFM images were recorded at different time points of insulin fibrillation as deducd from ThT fluorescence profile.

<p><b>A</b>) 0 h showing no remarkable structure for native insulin, <b>B</b>) 12 h, spherical shapes as oligomer structures, <b>C</b>) 22 h, single strand proto-fibrils, <b>D</b>) 30 h, long and mature fibrils with visible nodes because of twisted proto-fibrils. The scale bar represents 500 nm.</p

ThT fluorescence of β-Lg (5 mg/mL) at pH 2 heated at 80°C.

<p>(A) In the absence and presence of Pb²⁺, curcumin and their mixture. (B) In the absence and presence of AFM1, curcumin and their mixture. Data represent the means ± SD of 3 independent measurements.</p

The effects of different forms of β-Lg fibrils on neurite complexity of differentiated PC12 cells.

<p>The criteria were quantified after incubation for 4 or 8 h. (A) primary neurites per cell and (B) percent of bipolar cells.(n = 3; *significantly different from control).</p