Bovine insulin

Recombinant Aβ1-42 (rPeptide, Athens, GA, USA, lyophilized from hexafluoroisopropanol and dissolved in 2 mM NaOH) was diluted in 10 mM phosphate buffer supplemented with 14 mM NaCl and 2.7 mM KCl (PBS pH 7.4) to a final concentration of 10 μM and added to a 96-well microtiter plate (Corning Incorporated, Corning, NY, USA). Stock solutions of LCOs (1.5 mM) and ThT (2 mM) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0.3 μM. The samples were incubated at 37°C in a Tecan Saphire² microplate reader (Tecan, Männerdorf, Switzerland) and the emission spectrum of each probe was collected every 30 minutes. The emission spectra for t-HTAA, q-HTAA and q-FTAA were collected between 470–640 nm with excitation wavelength 430 nm. The emission spectra for p-HTAA and p-FTAA were collected between 480–650 nm with excitation wavelength 450 nm. The emission spectra for hx-HTAA, hx-FTAA, h-HTAA and h-FTAA were collected between 500–670 nm with excitation wavelength 480 nm. ThT was excited at 430 nm (emission between 470–640 nm) when used as a reference for the tetramers and it was excited at 450 nm (emission between 480–650 nm) when used as a reference for the pentamers, hexamers and heptamers. All samples were done in triplicates. Experiments replacing the Aβ 1-42 peptide with 10 μM bovine serum albumin (MP Biomedicals, Solon, USA) or 100 μM bovine insulin (I6634, Sigma Aldrich) were also performed in a similar fashion.
Excitation spectra for the LCOs were obtained at time point 0 minutes (right after the addition of soluble Aβ1-42) and for probes binding to Aβ 1-42 fibrils (at the plateau phase). The excitation spectra for t-HTAA (emission at 490 nm), q-HTAA (emission at 490 nm), q-FTAA (emission at 505 nm) and p-HTAA (emission at 490 nm) were collected between 270–470 nm. The excitation spectra for p-FTAA (emission at 507 nm), hx-HTAA (emission at 522 nm), hx-FTAA (emission at 535 nm), h-HTAA (emission at 538 nm) and h-FTAA (emission at 548 nm) were collected between 300–500, 310–510, 325–525, 335–535 nm, respectively.
Emission spectrum and excitation spectrum for each probe in PBS were also obtained using the same settings as described above.
Amyloid fibrils of bovine insulin were prepared according to a previously reported protocol^{29 (link), 38} . In brief, a stock solution containing 5 mg mL⁻¹ bovine insulin (I6634, Sigma Aldrich) in 2 M acetic acid supplemented with 500 mM NaCl was placed in a water bath kept at 50 °C to induce formation of amyloid-like insulin fibrils. Aliquots of 100 μL were withdrawn at different time points and mixed with 2 μL of the LCO stock solution (15 μM) or 2 μL of a ThT stock solution (15 μM). The fibrillation was also performed having 300 nM of LCO present during the fibrillation event. The samples were prepared in micro-titer plates and incubated for 10 min at room temperature. The emission spectra were recorded as described above.

Tumours were dissected from prostates of Pten^loxp/loxp Smad4^loxp/loxp PB-Cre4⁺ (Pten^pc−/−Smad4^pc−/−) mice, minced, and digested with 0.5% type I collagenase (Invitrogen) as described previously. After filtering through a 40-μm mesh, the trapped fragments were plated in tissue culture dishes coated with type I collagen (BD Pharmingen). Cells with typical epithelial morphology were collected, and single cells were seeded into each well of a 96-well plate. Three independent cell lines (Pten^pc−/−Smad4^pc−/−-1, -2 and -3,) were established and maintained in DMEM plus 10% fetal bovine serum (FBS, Omega Scientific), 25 μg ml⁻¹ bovine pituitary extract, 5 μg ml⁻¹ bovine insulin, and 6 ng ml⁻¹ recombinant human epidermal growth factor (Sigma-Aldrich). The prostate tumour epithelial cells express epithelial marker CK8 detected by immunofluorescence analyses using CK8 (GTX15465, GeneTex) antibody.