thioflavin-T (ThT) fluorescence assay

2.5. Measurement of intracellular reactive oxygen species (ROS) formation

Free radical production was measured by incubating the cells
with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH2-DA, Molecular Probe, USA). The DCFH2-DA dye, which is oxidized
to fluorescent 2′,7′-dichlorofluorescein (DCF) by hydroperoxide,
was used to measure the relative levels of cellular peroxides [34].


SH-SY5Y cells were pre-incubated in a 24-well culture plate (4×105
cells per well in 400 μL medium) for 24 h. Subsequently, cells were
rinsed by PBS, treated with each compound in serum-free medium,
and then incubated at 37 °C for 24 h.

After incubation, cells were treated with 400 μM H2O2 
for different times (5, 10, 15 and 20 min) and
then each well was rinsed with standard medium
(138 mM NaCl, 2.7 mMKCl, 1.2 mMCaCl2, 1.2 mMMgCl2, 10 mMPBS and 10 mMglucose, pH 7.4).

Following this, cells were further treated with 5 μM
DCFH2-DA for 30 min at 37 °C into standard medium. Cells were then
washed once with standard medium before measurement. Fluorescence
was measured at 485 nm excitation and 535 nm emission wavelengths
by aMicroplate Fluorescence Reader (SpectraMAX GEMINI EM,
Molecular Devices).


2.6. Thioflavin T fluorescence assay

Thioflavin T (ThT) fluorescence assay was performed with synthetic
Aβ1–42 (Bachem, Switzerland) as described previously [35].

The Aβ1–42 solution in PBS (50 mM NaH2PO4, 100 mM NaCl, 0.02%
NaN3) in a final concentration of 25 μM was incubated at 37 °C for
48 h to be aggregated in the presence or absence of each compound.


After incubation, a final concentration of 15 μM ThT was added to
each well containing the aggregated Aβ fibril, followed by additional
15 min of incubation at 37 °C.


ThT fluorescence was measured at 450 nm excitation and 490 nm emission wavelengths by a Microplate Fluorescence Reader
(SpectraMAX GEMINI EM, Molecular Devices).


Readings were normalized by the DMSO-only negative control and
the results were expressed as percentage of relative fluorescence to
the control. Tannic acid, previously shown to inhibit Aβ aggregation,
was used as a positive control [36].



2.7. Atomic force microscopy (AFM) measurements

The Aβ1–42 solution in PBS in a final concentration of 12.5 μM was
freshly prepared and aliquoted. Aβ1–42 peptides exist as monomers
when they are freshly prepared. Oligomerized Aβ1–42 was prepared
according to the method described in the cell viability assay. The fibril
form of Aβ1–42 was formed by incubation at 37 °C for 48 h. During the
oligomerization process, 20 μM of each compound was co-incubated
to check the ability of compounds in blocking Aβ1–42 oligomerization.
20 μL of each aliquot of monomer, oligomer, fibril Aβ1–42 sample solution
and oligomerized Aβ1–42 in the presence of each compound was
placed on the mica sheet (Structure Probe Inc., USA) and dried.

After this, the samples were scanned by AFM (XE-70, Park Systems, Korea),
and analyzed by XEI (Park Systems, Korea).


The reading scale of AFM micrograph was 4 μm×4 μm square,
composed of 256×256 pixels.
Each micrograph was measured with 256 maximum heights from
each horizontal line per pixel and average maximum heights were
obtained.

Oligomerization inhibition percentage was calculated from
the average maximum heights of oligomerized Aβ1–42 in the presence
of each compound vs oligomerized Aβ1–42 only.


2.8. Western blotting analysis
Cells and rat brain tissue were lysed by lysis buffer solution
containing 50 mMTris–HCl, 300 mMNaCl, 1% Triton X-100, 10% glycerol,
1.5 mM MgCl2, 1 mMCaCl2, 1 mM phenylmethanesulfonyl fluoride
(PMSF) and 1% protease inhibitor cocktail.

Protein concentrations of soluble extracts in the lysates were determined
by BCATM Protein Assay Kit (Pierce, USA).


A total of 50 μg of protein was loaded per well.
Samples were run on 12% polyacrylamide gels and transferred to
polyvinylidene fluoride (PVDF) membranes (Millipore Corporation,
USA). Membranes were blocked for 1 h at room temperature in
Tris-buffered saline with 0.1% Tween-20 (TBST) with 5% skim milk.
Membranes were incubated overnight at 4 °C with α-tubulin (1:1000
dilution, loading control), caspase-3 (1:1000), p35/25 (1:1000), MAPK
or p-MAPK (1:1000), tau or p-tau (1:1000) primary antibodies diluted
in TBST with 5% bovine serum albumin (BSA). Secondary antibodies
used were anti-mouse IgG horseradish peroxidase for tau and p-tau,
and anti-rabbit IgG horseradish peroxidase for α-tubulin, caspase-3,
MAPK, p-MAPK, and p35/25 antibodies. Secondary antibodies were
diluted (1:2000) in TBST with 5% skim milk. All antibodies were purchased
from Cell Signaling Technology Inc (USA). After 2 h of incubation
with secondary antibodies, membranes were washed three times
with TBST and then detected by ECL plus western blotting detection
reagent (Ab frontier, Korea). Western blot images were analyzed
using Multi-Gauge Software (Fuji photo film Co. Ltd., Japan).


2.9. μ-Calpain assay in cells
μ-Calpain assay in cells was performed as described previously [29].
In order to measure μ-calpain inhibitory activity of compounds in
SH-SY5Y and HEK293T cells, the human CAPN1 gene, which encodes
the μ-calpain catalytic subunit, was synthesized (Bioneer, Korea) [27].
The plasmid of CAPN1-encoding pcDNA3.1/His A was subcloned using
restriction sites EcoR V and Xho I, and was transfected into SH-SY5Y
and HEK293T cells using WelFect QTM Plus (Welgene, Korea). After
transfection for adequate times, cells were incubated with each compound
for 210 min in μ-calpain reaction buffer containing 0.1% Triton
X-100. The cleavage product of pep1 by μ-calpainwas measured in a kinetic
mode by a Microplate Fluorescence Reader (SpectraMAX GEMINI
EM, Molecular Devices) maintained at 37 °C. Fluorescence was measured
at 320 nm excitation and 420 nm emission wavelengths.
2.10. Immunocytochemistry