Supplementary Figures – Voccoli et al., 2014
(a)
10% FBS
Cell Viability (A.U.)
1.0
0% FBS
**
0.5
MBP
Tubulin
0.0
10% FBS
0% FBS
10% FBS
0% FBS
(b)
Figure S1. MO3.13 cell line characterization. (a) Cell viability quantification in case of cell
starvation (0% FBS) and culture maintenance condition (10% FBS), at t = 24 h. (n=9, MannWhitney test). (b) MBP expression increase upon starvation: (top) representative confocal
fluorescence images of MO3.13 cells immunostained for MBP (green) and stained with
Hoechst 33342 (blue); (bottom) Western Blot quantification of MBP expression (see
Materials and Methods for details, Sec. 5.4). Tubulin was used as loading control. Scale Bar =
100 μm.
Equation
y=y0+(A/(w1*sqrt(PI/2)))*exp(-2*((x-xc1)/w1)^2
)+(B/(w2*sqrt(PI/2)))*exp(-2*((x-xc2)/w2)^2)
16
14
Adj. R-Square
0,8526
Value
12
Count
10
8
Standard Error
Count
y0
-0,29128
0,57775
Count
A
857,75885
85,67424
Count
B
539,13243
177,53391
Count
w1
49,52808
4,55877
Count
w2
106,58075
29,53136
Count
xc1
35,22272
2,24945
Count
xc2
165,21668
10,96025
200
250
6
4
2
0
0
50
100
150
300
350
time (min)
Figure S2: MO3.13 Lifetime distribution measured during time-lapse experiments upon
PSY 10 μm administration (t=0): histogram of cell-death events as a function of time (10-min
binning). The histogram has been fitted with a double Gaussian curve (black line); the best
fitting parameters are reported in the table.
(a)
1.2
0.8
0.8
Fluo-3
TMRM
DMSO
0.4
0.4
0
TMRM (A.U.)
Fluo-3 (A.U.)
1.2
0
100
200
300
400
0
Time (min)
(b)
2.2
1.2
0.8
TMRM (A.U.)
MitoCa2+ (A.U.)
1.2
MitoCa 2+
TMRM
0.4
0.2
DMSO
0
0
100
200
300
400
Time (min)
(c)
1,2
0.8
DMSO
0.4
Fluo-3
MitoROS
0
0
100
200
300
0.4
MitoROS (A.U.)
Fluo-3 (A.U.)
0.8
0
400
Time (min)
Figure S3. Representative kinetics control experiments. Representative traces of (a)
cytoplasmic Ca2+ (green line) and mitochondrial potential (red line), (b) mitochondrial Ca2+
(green line) and mitochondrial potential (red line), and (c) cytoplasmic Ca2+ (green line) and
mitochondrial ROS (yellow line) upon vehicle (DMSO) administration (blue arrow).
Figure S4. MTCD2CPV localization. MO3.13 cells transfected with the mitochondrial Ca2+
probe MTCD2CPV and stained with TMRM were imaged along the Z-axis every 0.2 μm with a
resolution of 1024x1024 pixels. The panel “ merge” shows the good colocalization (yellow) of
the MTCD2CPV with mitochondria (TMRM signal). Cells were rebuilt with MetaMorph 5.0
software (Universal Imaging, West Chester, PA) in Z-stacks. After background subtraction a 2
pixel size median filter was applied to the stacks. The 3D rendering of the colocalization map
superimposed to the cell bright field image was performed by using Amira software (bottom
row). Scale Bar = 20 μm.
**
**
0.8
M
ED
TA
5
m
M
m
3
ED
TA
1m
ED
TA
FB
%
(0
U
nt
re
at
ed
M
0.6
S)
Cell viability (A.U.)
NS
1.0
Figure S5. EDTA dose response quantification of cell viability. MO3.13 cells were treated
in 0% FBS with EDTA 1mM, 3mM and 5mM for 24 hours and analyzed by AV/PI flow
cytometry [n = 3, One-Way ANOVA (Dunnett’s post-hoc test) EDTA 1-5 mM vs Untreated].
(a)
PSY 10 mM
PSY + NAC
Mitochondrial ROS
PSY + NAC
Annexin V
(d)
1.5
Mitochondrial ROS (A.U.)
PSY 10 mM
3
**
1.0
NS
*
*
0.5
2
NS
NS
1
0
0.0
3 mM
PSY
NAC
Cell viability (A.U.)
(b)
Propidium Iodide
SSC
(c)
-
5 mM
+
-
10 mM
+
-
3 mM
PSY
+
NAC
-
5 mM
+
-
10 mM
+
-
+
Figure S6. Effect of NAC on Cell Viability and mitochondrial ROS production. (a)
Representative dot-plots of MO3.13 cells treated with PSY 10 M or with PSY 10 M in
presence of NAC 5mM, stained with Mitotracker Red CMXRos and analysed by flow cytometry.
The side-scattered light signal (SSC) is also reported. The R6 region was set by the dot-plot
obtained for the control population in (No PSY, 10% FBS). (b) Mitochondrial ROS production
quantification of the flow-cytometry dot-plots for cells treated with PSY 3–10 M, with (+) of
without (-) NAC 5 mM administration. [n>3, t-test NAC (-) vs NAC (+) for same PSY
concentrations]. (c) Representative dot-plots of MO3.13 cells treated with PSY 10 M or with
PSY 10 M in presence of NAC 5mM, stained with Annexin V/Propidium Iodide and analysed
by flow cytometry: R8, R9, R7 and R3 quadrants define the healthy, apoptotic, secondary
necrotic and necrotic populations, respectively. (d) Cell viability quantification of the flowcytometry dot-plots for cells treated with PSY 3–10 M, with (+) of without (-) NAC 5 mM
administration. [n>3, t-test NAC (-) vs NAC (+) for PSY 3 M and 10 M; Mann-Whitney test
NAC (-) vs NAC (+) for PSY 5 M].
(b)
1.0
0.5
C
C
A
+N
A
ED
TA
%
(0
ed
N
FB
A
C
ED
TA
0.0
TA
+N
C
A
N
TA
ED
NS
re
nt
U
U
nt
re
at
ed
(0
%
FB
S)
0.6
NS
NS
at
Cell viability (A.U.)
0.8
1.5
ED
NS
1.0
S)
NS
NS
Mitochondrial ROS (A.U.)
(a)
Figure S7. EDTA 1mM and NAC 5mM do not affect cell viability and mitochondrial ROS
production. MO3.13 cells treated with EDTA 1 mM, NAC 5 mM and EDTA 1 mM + NAC 5 mM,
stained with Mitotracker Red CMXRos or AV/PI, and analyzed by flow cytometry: (a)
mitochondrial ROS production quantification [4 < n < 7, One-Way ANOVA (Dunnet’s post-hoc
test EDTA, NAC, EDTA+NAC vs Untreated] and (b) cell viability quantification [n = 3, One-Way
ANOVA (Dunnet’s post-hoc test EDTA, NAC, EDTA+NAC vs Untreated].
1.2
TMRM (A.U.)
1.0
0.8
0.6
0.4
CCCP (25 mM)
Vehicle
0.2
0.0
-20
0
20
40
Time (min)
Figure S8. TMRM fluorescence upon CCCP administration. Representative traces of
MO3.13 single-cell TMRM fluorescence kinetics upon CCCP (25 μM, black) and vehicle (gray)
administration (t = 0; black arrow). Data from 8 (n=3) and 3 (n=1) cells from independent
experiments were collected in single datasets and single-cell fluorescence averaged values
were reported with standard deviations as error bars. CCCP was dissolved in EtOH (at 25
mM) and added into Willco dishes to obtain a solvent final concentration of 0.1% in the cell
medium.