Fig 1.
SEC–HPLC chromatograms of amyloids preparation.
(A)–Aβ(1–42)–HFIP protocol preparation; (B)–Aβ(1–42)–HFIP-free protocol preparation. The sample concentration was 10 μM. Mobile phase– 0.1 M NaCl, 0.01 M NaH2PO4 buffer at pH 7.4 and flow rate of 0.25 ml/min.
Fig 2.
Representative AFM images of adsorbed 10 μM Aβ(1–42) oligomers prepared by HFIP protocol (A) and HFIP-free protocol (B). Mica surfaces (1 μm2) were visualized after 10 min incubation with preparations.
Fig 3.
The actual height and Gauss approximation distributions of Aβ(1–42) adsorbed on mica.
HFIP-protocol (black box and line) and HFIP-free protocol (box with diagonal lines and dashed line).
Fig 4.
FTIR spectra of Aβ(1–42) oligomers of different size.
FTIR spectra of HFIP protocol (a) and HFIP-free protocol (b) Aβ(1–42) oligomers deposited at CaF2 substrate in the spectral region of 1200−1800 cm−1.
Fig 5.
Comparison of Aβ(1–42) oligomers in Amide-I spectral region.
FTIR absorption spectra with fitted Gaussian-Lorentzian form components in Amide-I spectral region: (A) spectra of Aβ(1–42)–HFIP protocol, and (B) Aβ(1–42)–HFIP-free protocol; both deposited at CaF2 substrate.
Fig 6.
Second derivative spectra of Amide-I band.
(a) Amide-I band of HFIP protocol, and (b) HFIP-free protocol of Aβ(1–42) oligomers deposited at CaF2 substrate in the spectral region of 1590−1760 cm−1.
Table 1.
Amide-I peak positions and integrated intensities with corresponding band assignments of Aβ(1–42) peptide.
Fig 7.
Evaluation of growth inhibition of cancer cell lines treated with amyloids.
The anti-proliferative activities of amyloids were tested using the MTT assay as previously described. (A)–growth inhibition of cells treated with HFIP protocol amyloids, concentration range 0.5–2 μM. (B)–growth inhibition of cells treated with HFIP-free protocol amyloids, concentration range 1–5 μM. P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***) indicate significant differences, if not indicated otherwise the difference was not significant.
Fig 8.
Distribution of cell cycle phases of cancer cells treated with amyloids.
Amyloid activity on cell cycle distribution was analyzed by flow cytometry, as detailed in the Materials and methods section. (A)–cell cycle of cells treated with 1 μM HFIP protocol amyloids and solvent for negative control. (B)–cell cycle of cells treated with 2–5 μM HFIP-free protocol amyloids and solvent for negative control.
Fig 9.
Induction of cancer cell death by amyloids.
The pathway of cell death induced by amyloids was analyzed by flow cytometry. Representative scatter plots showing Annexin V and PI staining of NB4 cells, I–necrosis (Annexin V-/PI+), II–late apoptosis (Annexin V+/PI+), III–early apoptosis (Annexin V+/PI-). (A)–NB4 cells treated with 2 μM of HFIP protocol amyloids and solvent for negative control. (B)–NB4 cancer cells treated with 5 μM of HFIP-free protocol amyloids and solvent for negative control.
Fig 10.
Fluorescence analysis of cancer cells with FAM-labeled amyloids.
Amyloid accumulation in cancer cells after certain periods of time (4, 24, 48 h) and control cells without amyloid treatment. FAM-labeled amyloids are shown in green, DAPI stained nuclei are shown in blue. Magnification x63.