6
K. PRIYADARSHI ET AL.
Caspase-3 activity assay
(Waghela et al., 2015). In brief, After completion of treatment,
cells were harvested and lysed in lysis buffer containing
The activity of caspase-3 was examined by using the EnzChek
Caspase-3 Assay Kit (Invitrogen, Life Technologies, USA)
2
50 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na HPO ,
2 4
1
.4 mM KH PO 100 lM PMSF and 1X protease inhibitor cock-
6
2
4,
according to the manufacturer’s protocol. Briefly, 1 ꢁ 10 HCT
tail (Roche, Germany) for 5 min on ice. The lysate was centri-
1
16 cells were treated with 2.5 mM, 5 mM and 10 mM of
ꢀ
fuged at 1,000 ꢁ g for 5 min at 4 C and the resulting
PAMAM-GA conjugate for different time points as shown in
the figure legend. Untreated cells were used as control. After
completion of incubation, cells were harvested and washed
twice with DPBS. The pellet was resuspended in 1X cell lysis
supernatant was collected as the cytosolic fraction. The pellet
was resuspended in mitochondrial lysis buffer containing
5
0 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM
EGTA, 0.2% (v/v) Triton X-100, 0.3% NP-40, 100 lM PMSF and
X protease inhibitor cocktail for 5 min on the ice. Pellet was
ꢀ
buffer for 30 min and centrifuged at 5000 rpm for 5 min at 4 C.
1
The amount of protein in the resultant supernatant was esti-
mated using the BCA protein estimation kit. The 100 mg pro-
tein was mixed with a 2X substrate solution containing 50 mM
Z-DEVD-R110 substrate and incubated at room temperature
for 30 min in the dark. The fluorescence was measured at an
excitation and emission wavelength of 496 nm and 520 nm
respectively using multimode plate reader (SpectraMax M2e,
Molecular Devices, USA).
disrupted in cell disruptor and centrifuged at 10,000 ꢁ g for
ꢀ
5
min at 4 C and the supernatant was collected as a mitochon-
drial fraction to examine the expression of cytochrome c and
COX-IV. The whole cell lysate was prepared using RIPA buffer
containing a protease inhibitor cocktail (Roche, USA). Cells
were kept on the ice for 30 min followed by disruption and
ꢀ
centrifugation at 8,000 rpm for 10 min at 4 C and supernatant
was collected. For collection of nuclear fractions, cells were
lysed in hypotonic buffer (10mM HEPES-Kþ pH 7.5, 10mM KCl,
1
1
.5mM MgCl2, 0.1mM DTT, 0.5% Triton X-100, 1mM PMSF and
Necrotic cell death assay
ꢀ
x PIC) for 5 min on ice and spun at 3000 rpm for 2 min at 4 C.
The Necrotic cell death (cytotoxic effect) of GA and PAMAM-
GA conjugate was examined by release of LDH using LDH
activity assay kit (Takara Bio Inc., Shiga, Japan) according to
the manufacturer’s instruction. Briefly, HCT 116 cells and NIH
The pellets were washed with wash buffer (10mM HEPES-Kþ
pH 7.5, 10mM KCl, 1.5mM MgCl2, 0.1mM DTT, 1mM PMSF and
1
1
x PIC) and subsequently lysed with lysis buffer (150mM NaCl,
% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50mM
3
T3 cells were treated with 2.5 mM, 5 mM and 10 mM of
Tris pH 8.0, 1mM PMSF, 1mM NaV and 1x PIC) for 25 min on
ice and spun at 10,000 rpm for 15 min. The supernatant was
collected as nuclear fraction. Protein concentration was deter-
mined by using a BCA protein estimation kit (Sigma-Aldrich,
USA) according to the manufacturer’s protocol. 50 lg protein
was resolved on 10% SDS-PAGE and transferred to PVDF mem-
PAMAM-GA conjugate for 6, 12 and 24 h. The assay was per-
formed as mentioned previously (Ranjan & Pathak, 2016).
Microscopic assessment of cytotoxicity was carried out using
Hoechst and propidium iodide staining as described previ-
ously (Vaidya et al., 2019). Cells were analyzed under a fluor-
escence inverted microscope (DP71, Olympus, Japan) and
images were analyzed using ImageJ software (NIH,
Bethesda). Hoechst-stained nuclei were counted and judged
as either viable (lack of propidium iodide staining), apoptotic
ꢀ
brane by wet electro-blotting method at 4 C. The membrane
was blocked with 5% non-fat milk in TBST (10 mM Tris-HCl (pH
7
.4), 0.9% NaCl and 0.05% Tween 20) for 4 h at room tempera-
ture followed by overnight incubation with primary antibody
against p65 (1:1000), cytochrome c (1:1000), procaspase-3
(
Hoechst-stained condensed nuclei with propidium iodide-
stained spots), or necrotic (large nuclei intensely stained with
Hoechst and propidium iodide-stained).
(
1:1000), PARP (1:1000), Bcl-2 (1:250), MMP-9 (1:250), Histone
H3 (1:1000), COX-IV (1:1000), b-actin (1:10,000) and GAPDH
ꢀ
(1:1000) at 4 C. After washing with TBST, the membrane was
probed with HRP conjugated secondary antibodies (1:10,000).
Expression of immune reactive protein was detected by using
Lysosomal integrity assay
The effect of GA and PAMAM-GA conjugate on lysosomal mem- Clarity Western ECL substrate kit according to the man-
brane integrity was examined by using LysoTracker Red DND- ufacturer’s instruction and developed on Kodak X-Omat blue
9
9 dye (Invitrogen, Life Technologies, USA). HCT 116 cells were film (NEN Life Sciences, Inc., Boston, MA) in the dark.
treated with GA and PAMAM-GA conjugate shown in the figure
ꢀ
legend and further stained with Lysotracker red for 1 h at 37 C,
Co-immunoprecipitation assay
followed by counterstaining with DAPI (1 mg/ml) for 5–7 min in
the dark. More than 100 cells from random fields were exam-
ined under a fluorescence inverted microscope (DP-71, IX81,
Olympus, Japan). All the images were acquired by Image-Pro
MC 6.1 (Bethesda, MD, USA) and analyzed by Image J
To analyze the formation of the apoptosome complex, a co-
immunoprecipitation assay was carried out as described ear-
6
lier (Ranjan & Pathak, 2016). In brief, 1 ꢁ 10 HCT 116 cells
were seeded in 60 mm dishes and treated with PAMAM-GA
conjugate for 12 h. The cells were lysed using lysis buffer
(
NIH, USA).
(20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 10% glycerol, 1%
Triton-X-100) containing 1X protease inhibitor cocktail
Sub-cellular fractionation and western blotting
ꢀ
(
Roche, USA) for 30 min at 4 C, followed by disruption and
ꢀ
The expression of cell death regulatory proteins was examined centrifugation at 15,000 ꢁ g for 15 min at 4 C. The clarified
from mitochondrial and cytosolic fractions as described earlier supernatant was collected and its protein concentration was