supplementary figure s1 cell index 1224364860728496 hours 1 2 3 4 5 6 cell index 1224364860728496...
DESCRIPTION
200 ABTL Vehicle A MiaPaca-2 A549 B STP0 cleaved Caspase 3 15 Actin PARP-1 cleaved PARP-1 A549 cellsMiaPaca-2 cells DEVDase activity (A.U.F/µg protein) STP0 STP 1µM Supplementary Figure S3 Supplementary Figure S3. ABTL0812 does not induce apoptotic cell death. (A) Cells were treated with the indicated doses of ABTL0812 or 1µM staurosporine (STP, as a positive control for apoptosis) for 24 h. Cells were fixed and nuclei stained with Hoechst. Cell nuclei were visualized with a Nikon ECLIPSE TE2000-E microscope equipped with epifluorescence optics. Representative pictures showing normal nuclear morphology of ABTL0812-treated cells, and apoptotic nuclear morphology (condensed or fragmented nuclei) in STP-treated cells. (B) Cells treated with the indicated amounts of ABTL0812 ( M) or 1 M STP for 24 h were lysed, and Caspase-3-like activity was measured using the fluorogenic Ac-DEVD-AFC reagent (histograms) in a Bio-Tek FL 600 Fluorometer. Caspase-3 activity was calculated as the increase in arbitrary fluorescence units per microgram of protein. Each value is the mean ± SD of three different determinations, each performed in triplicate, and normalized per microgram of protein. Cells were also analyzed for capase-3 activation by monitoring levels of the cleaved caspase-3 and PPAR-1 by immunoblotting (lower panels). Actin was used as loading control.TRANSCRIPT
Supplementary Figure S1
Cel
l in
dex
12 24 36 48 60 72 84 96 hours
1
2
3
4
5
6
Cel
l in
dex
12 24 36 48 60 72 84 96 hours
1
2
3
4MiaPaca-2 cells
vehicle
vehicle
ABTL0812
A549 cells
ABTL0812
Supplementary Figure S1. ABTL0812 inhibits cell proliferation.
A549 and MiaPaca-2 cells, seeded in L8 E-Plates, were treated with either vehicle (blue circles and line)
or 50 mM ABTL0812 (red circles and line). Impedance, which correlates with plate surface covered by
attached cells, was monitored in real time during 96 h on a xCELLigence System (Acea Biosciences).
Electronic readout of cell-sensor-detected impedance is displayed as arbitrary units called Cell Index.
Arrows denote the time when vehicle or ABTL0812 were added to the cells. Each value is the mean ± SD
of three different determinations performed in parallel experiments.
FL3 Log (PI fluorescence)
Coun
tsVehicle
ABTL0812
A549 cells MiaPaca-2 cells600
400
200
0
400
200
010 102 1031 10 102 1031
Coun
ts
40
20
010 102 1031 10 102 1031
50
25
0
Coun
ts
Coun
ts
Vehicle
ABTL0812
FL3 Log (PI fluorescence)
Supplementary Figure S2
Supplementary Figure S2. ABTL0812 induces cell death.A549 and MiaPaca-2 cells were treated with either vehicle or 100 mM ABTL0812 for 24 h. Cells were collected and incubated with 1 mg/ml propidium iodide (PI), and analyzed using flow cytometry. Similar results were obtained in three separate experiments. Lower histograms show percentage of death cells (PI positive). ***p<0.001 from vehicle-treated cells
A549 cells MiaPaca-2 cells
20
40
60
80
0
% c
ell d
eath
( P
ropi
dium
iodi
de-p
ositi
ve c
ells) vehicle
ABTL0812******
200ABTL-0812
400 600VehicleA
Mia
Paca
-2A5
49
B
200 400 600
100
STP0
cleaved Caspase 315
Actin
PARP-1 cleaved PARP-1
A549 cells MiaPaca-2 cells
DE
VD
ase
activ
ity
(A.U
.F/µ
g pr
otei
n)
100
200
300
400
25
50
75
100
125
200 400 600 STP0
STP 1µM STP 1µM
Supplementary Figure S3
Supplementary Figure S3. ABTL0812 does not induce apoptotic cell death.(A) Cells were treated with the indicated doses of ABTL0812 or 1µM staurosporine (STP, as a positive control for apoptosis) for 24 h. Cells were fixed and nuclei stained with Hoechst. Cell nuclei were visualized with a Nikon ECLIPSE TE2000-E microscope equipped with epifluorescence optics. Representative pictures showing normal nuclear morphology of ABTL0812-treated cells, and apoptotic nuclear morphology (condensed or fragmented nuclei) in STP-treated cells. (B) Cells treated with the indicated amounts of ABTL0812 (mM) or 1 mM STP for 24 h were lysed, and Caspase-3-like activity was measured using the fluorogenic Ac-DEVD-AFC reagent (histograms) in a Bio-Tek FL 600 Fluorometer. Caspase-3 activity was calculated as the increase in arbitrary fluorescence units per microgram of protein. Each value is the mean ± SD of three different determinations, each performed in triplicate, and normalized per microgram of protein. Cells were also analyzed for capase-3 activation by monitoring levels of the cleaved caspase-3 and PPAR-1 by immunoblotting (lower panels). Actin was used as loading control.
Mia
PaC
a-2
A54
9Vehicle ABTL0812
Supplementary Figure S3. ABTL0812 induces LC3 association with autophagosomes.Cells were treated with vehicle (ethanol) or 50 mM ABTL0812 for 12 h, before immunostaining for endogenous LC3 (green) and nuclei (Hoechst, blue). Punctua represent autophagosome formation.
Supplementary Figure S4
PPARα PPAR
0.1 1 10 100 1000
25
50
75
100
Concentration ABTL0812 (mM)
IC50: 6.8 µMKi: 7.1 µM
% R
adio
ligan
d b
indi
ng
0.1 1 10 100 1000
25
50
75
100
Concentration ABTL0812 (mM)
IC50: 2.5 µMKi: 4.7 µM
% R
adio
ligan
d b
indi
ng
Supplementary Figure S5
Supplementary Figure S5. ABTL0812 binds PPARα and PPAR ligand binding domains
Purified human recombinant PPARα or PPAR were incubated with ABTL0812 and labeled PPARa
agonist 3H-WY14643 (200 nM) or PPAR agonist 3H-Rosiglitazone (5nM). Values are the mean ±
SD of two separate determinations performed in duplicate. Ki values were calculated using the
Cheng-Prussoff equation (7).
A549 Cells MiaPaca-2 cells
Supplementary Figure S6
PP
RE
-Luc
iera
se a
ctiv
ity (f
old
indu
ctio
n)
1
3
5
7
vehic
le
WY-
1463
Rosigl
itazo
ne
ABTL081
2
vehic
le
WY-
1463
Rosigl
itazo
ne
ABTL081
2
1
3
5
7
PP
RE
-Luc
iera
se a
ctiv
ity (f
old
indu
ctio
n) ***
***
*** ****** ***
Supplementary Figure S6. ABTL0812 induces PPRAa and PPAR transcriptional activities.
Luciferase-PPAR response element (PPRE) reporter and pRL-CMV-Renilla plasmids were transiently
transfected in A549 and MiaPaca-2 cells. 16 h post-transfection, cells were treated with vehicle, PPARa
agonist WY-14643 (1 mM) or PPAR agonist rosiglitazone (1 mM) or 50µM ABTL0812. After 24 h, lysates
were subjected to a dual-luciferase reporter assay. Each value is the mean ± SD of three different
experiments, each performed in triplicate, and normalized using the Renilla values. ***P<0.001 from
cells treated with vehicle.
0.0
0.5
1.0
1.5
2.0
2.5
TRIB
3 m
RN
A le
vels
(fol
d fro
m v
ehic
le o
f sam
ple
1)
VehicleABTL0812
Sample 1
Sample 2
Sample 3
Sample 4
0.0
0.5
1.0
1.5
2.0 **
GAPDH
TRIB3
S1 S2- +
Vehicle ABTL
- +
Supplementary Figure S7
Supplementary Figure S7. ABTL0812 induces TRIB3 mRNA levels in Peripheral blood
mononuclear cells (PBMC). PBMCs purified from four different human samples were treated for
6 h with vehicle (white columns) or 100 mM ABTL0812 (blue columns). After purifying total RNAs
with Total RNA Mini Kit Blood/Cultured Cell (Geneaid, New Taipei City, Taiwan), TRIB3 mRNA levels
were monitored by RT-qPCR. Value of sample 1 was arbitrarily set to 1. Inset upper panel: data
correspond to the mean ± SD of samples 1 to 4, and it shows the mean fold change relative to
non-treated cells; (n=4;**P < 0.01 from non-treated cells). Inset lower panel: TRIB3 mRNA levels
(as determined by RT-PCR) from ABTL-0812-treated (+) or non-treated (-) samples 1 and 2.
ABTL
TRIB
3 m
RN
A le
vels
(fol
d fro
m v
ehic
le)