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Supplemental materials and methods
LDH and MTT assays.
N2a cells were seeded in 96-well plates at a density of 50,000 cells/100 l per well.
Twenty fours after cell seeding, N2a cells were transiently transfected with
pcDNA-14-3-3 isoform plasmids by lipofection. Twenty fours after transfection, N2a
cells were subjected to 2 h of OGD incubation. Cell death/viability was determined by
the
release
of
lactate
dehydrogenase1
(LDH;
Promega,
USA)
or
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide2 (MTT; Sigma, USA)
assay as reported previously. Optical absorbance at 492 nm was measured by a
microtiter plate reader (Synergy 2 BioTek, USA). Maximal LDH release was obtained
by cell lysis with DMSO and relative LDH release was calculated according to the
formula provided by the manufacturers.
Primary cultures of rat cerebral cortical astrocytes.
Primary cultured astrocytes were prepared from cerebral cortices of newborn SD rats
as reported previously. The culture media was changed after 2 days of initial seeding
and subsequently twice per week with DMEM containing 10% FBS. The cultures
were confluence around 10-14 days. Astrocytic cultures were used at 4 weeks old.
CoCl2 treatment.
Adult male Kun-ming mice (22±2 g) were purchased from the Animal Center of
Tongji Medical College, Huazhong University of Science and Technology (HUST),
China, and housed in a 12:12 h light-dark cycled room maintained at 22±2°C with
food and water ad libitum for 5 days prior to experiments. CoCl2 was used to induce
ischemic pre-conditioning as reported1-2. Mice were randomly divided into normal
saline (NS) and CoCl2 groups and administered with CoCl2 (Sigma, USA) by
intraperitoneal injection (i.p., 30 mg/kg). Each mouse was placed into a 125-ml jar 4 h
after injection with fresh air containing 8 g of soda lime. The jar was sealed with a
rubber plug to induce hypoxic hypoxia3-4. After the appearance of quick respiration,
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increasing cyanosis, spasm-like activity and grasping, breathing stopped. Immediately
after breathe stop, the animals were sacrificed and total proteins were extracted from
the cerebral cortices of mice for Western blot analysis.
Terminal Deoxynucleotidyl Transferase End-labeling (TUNEL).
After the incubation of primary and fluorescent secondary antibodies, brain sections
or cultured neurons were washed three times with PBS and then permeabilized with
freshly prepared PBS with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on
ice. TUNEL staining was performed following the manufacturer’s instructions (In Situ
Cell Death Detection Kit, TMR red, Roche Diagnostics, Germany). Micrographs were
taken with a Zeiss 510 confocal microscope (Carl Zeiss, Germany).
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Supplemental Figures
Figure S1
isoform
Representative Western blot results showing the specificity of 14-3-3
antibodies.
N2a
cells
were
transiently
transfected
with
pcDNA-14-3-3 and  for 2 d. Equal amount of total protein extracted
from transfected N2a cells were subjected to Western blot analysis with
isoform-specific 14-3-3 antibodies (and  Mo, mouse; Rb, rabbit. -actin
was used as internal control.
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Figure S2
Representative micrographs of IHC showing the expression of 14-3-3
isoforms in ischemic brains. Brain slice from 1 h-pMCAo rats were subjected to
immunohistochemistry (IHC) analysis with 14-3-3 or antibodies. Cotra,
contralateral cerebral cortex; Ipsi, ipsilateral cerebral cortex.
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Figure S3
Representative micrographs of fluorescent double-immunostaining
showing the relationships between 14-3-3 levels and cell death or survival in
cultured neurons 24 h post 2 h-OGD. Primary cultures of rat cerebral cortical
neurons subjected to 2 h of OGD were replaced with normal culture media and
incubated under normal culture conditions for another 24 h. Cultured neurons were
stained with 14-3-3/cleaved caspase 3/Hoechst 33342 (upper panels) or
14-3-3/TUNEL (lower panels) simultaneously.
Conclaved arrowheads indicated
elevated 14-3-3 in surviving neurons while arrows indicated decreased 14-3-3 in
apoptotic neurons.
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Figure S4
Statistical analysis of the effects of 14-3-3 isoform overexpression on
cell death in N2a cells upon OGD. N2a cells were transiently transfected with
pcDNA-14-3-3or  for 24 h and subjected to 2 h of OGD incubation. (a)
Relative LDH release. LDH release in 14-3-3 isoform-transfected N2a cells upon 2 h
of OGD was measured using the LDH assay kit. Relative LDH level was normalized
to that of maximal level by cell lysis of the culture. **P<0.01 vs. pcDNA (n=3). (b)
MTT assay. Cell viability in 14-3-3 isoform-transfected N2a cells upon 2 h of OGD
was measured by using MTT. Relative viability was normalized to that of 0 h control.
*P<0.05 vs. Vec (n=3).
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Figure S5
Effects of 14-3-3 isoforms on Bax expression and Bax overexpression on
cell death in N2a cells. (a) Representative Western blot results and statistical analysis
showing the effects of 14-3-3 isoforms on Bax expression. N2a cell were transiently
transfected with pcDNA-14-3-3 or for 2 d. -catin was used as an
internal control. Relative Bax level was normalized to -actin level. *P<0.05,
**P<0.01 vs. Vec (pcDNA) (n=3). (b) Representative fluorescent micrographs and
statistical analysis showing the effect of Bax overexpression on cell death in N2a cells.
N2a cells were transiently co-transfected with equal amount of p-EGFP-N1 with
either pcDNA or pcDNA-Bax (provided by Dr. Yan Zhang, Peking University) at a
ratio of 1:1 for 2 d. Arrows indicated dead cells. The fluorescent intensities of GFP
were measured randomly at 9 fields in each culture with PLUS software and the
average GFP fluorescence represented cell viability of co-transfected cells. ***
P<0.001 vs GFP+pcDNA (n=3).
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Figure S6
Early induction of 14-3-3 upon ischemic insults. (a) Statistical analysis
of 14-3-3 levels in the contra and Ipsi during pMCAo. Brain slice from rats subjected
to various time of pMCAo (1 h, 3 h, 6 h, 1 d, 7 d or 20 d-pMCAo) were used for
analyzing 14-3-3by IHC. Relative intensity of 14-3-3 from more than 100 neurons
in each IHC micrograph was measured by using the Plus software. Relative
14-3-3levels in the Ipsi were compared to that of Contra. * P<0.05, **P<0.01 vs
Contra (n=7). (b) Representative Western blot results and statistical analysis of
14-3-3 expression in cerebral cortex of newborn rats after decapitation. New-born
rats were decapitated for 0, 15 min, 30 min, 1 h or 2 h and total protein were extracted
from cerebral cortex of mouse brain. Relative 14-3-3/-actin level was compared to
that of 0 h. *P<0.05, **P<0.01 vs 0 h (n=5). (c) Representative Western blot results
and statistical analysis of 14-3-3 levels in cultured cortical neurons upon hypoxia.
Cultured neurons at 7 DIV were exposed to 0, 1, 3, 6 or 12 h of hypoxia (0.1% O2).
Relative 14-3-3/-actin level was compared to that of 0 h. *P<0.05, **P<0.01 vs 0 h
(n=3). (d) Representative Western blot results and statistical analysis of 14-3-3 levels
in cultured cortical neurons upon serum and glucose deprivation. Cultured neurons at
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7 DIV were incubated with DMEM free of serum and glucose for 0, 3, 6, 9, 12 or 24 h.
Relative 14-3-3 level (14-3-3/-actin) was compared to that of 0 h. *P<0.05,
**P<0.01 vs 0 h (n=3).
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Figure S7
Selective induction of 14-3-3 by CoCl2 in cerebral cortex of mice. (a)
Representative Western blot results. Adult mice were administrated with CoCl2 for 4 h
via i.p. injection. Western blot analysis was performed with total proteins extracted
from the cerebral cortex of mouse. 1-5 indicated the number of mouse in each group.
Normal saline (NS) was used as the vehicle control. (b) Statistical analysis of relative
levels of 14-3-3 isoforms in mouse cerebral cortex pre-treated with CoCl2. **P<0.01
vs NS. (n=5).
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Figure S8
Representative micrographs of fluorescent double-immunostaining
showing the relationships between 14-3-3 levels and cell death or survival in
ischemic rat brain subjected to 24 h-pMCAo. Conclaved arrowheads indicated
elevated 14-3-3 in survived neurons while arrows indicated decreased 14-3-3 in
apoptotic cells.
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Figure S9
Proposed protective mechanism of 14-3-3γ in ischemic neurons and
astrocytes. 14-3-3γ was elevated in both astrocytes and neurons under ischemia. In
neurons, elevated 14-3-3γ bound to more p-β-catenin Ser37 to suppress Bax
expression. In astrocytes, elevated 14-3-3γ bound to more p-Bad Ser112 to block the
apoptotic effect of Bad.
, induction or activation;
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, binding;
, inhibition.
Figure S10 14-3-3 does not bind to p--catenin Ser37 or alter Bax expression in
primary cultured cortical astrocytes upon OGD. Primary cultures of rat cerebral
cortical astrocytes at 4 weeks were subjected to 0, 2 or 4 h of OGD. (a)
Representative Co-IP results. Four hundreds of total soluble proteins from
OGD-treated astrocytes were incubated to 1 g of mouse anti-14-3-3 antibodies.
Equal amounts of immunoprecipitates were subjected to Western blot analysis with
rabbit anti-p--catenin Ser37, -catenin or 14-3-3 antibodies. Mouse IgG was used a
negative control for co-IP. WCL, whole cell lysate. (b) Representative Western blot
results.
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Figure S11
(a) Physiologic parameters in SD rats subjected to pMCAo. (b)
Representative micrographs showed the regional cerebral blood flow monitored
during pMCAo by a Laser Doppler Perfusion Monitor. A sharp decrease of cerebral
blood flow to lower than 20% of the baseline indicated the success of MCAo.
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