Supplementary Materials and Methods
Lentivirus package
Lentivirus particles were produced by calcium phosphate transfection according to the
method previously described.1 Briefly, 293FT cells (Invitrogen) were cultured and
co-transfected with generated lentiviral vectors and two other helper vectors. The
medium was replaced 12 hrs later. The supernatant was harvested 48 hrs and 72 hrs
after transfection and then concentrated by precipitation with PEG-8000 (Sigma). The
resulting pellet was resuspended, aliquoted and frozen at -80°C until use.
Primary VM neurons and primary glia cells
Primary VM neurons were prepared as described previously.2, 3 Briefly, the
ventral midbrain tissues were carefully dissected from the fetuses of E13.5
Sprague-Dawley rats, dissociated mechanically and digested by 0.25% trypsin and
0.01% DNase. The VM neurons were collected by centrifugation and finally cultured
on poly-D-lysine (PDL)-coated 96-well plates at a density of 1.6×105/cm2 with
complete DMEM/F12 (DF12) medium (Gibco). The cells were incubated at 37°C in a
humidified 5% CO2 atmosphere for 12 hrs and then replaced with serum free DF12
medium, with the addition of 2% B27 supplement (Gibco), and incubated with
medium change every 3 days. One week later, VM neurons were cultured with CM
from treated microglia cells for 24 hrs followed by survival assessment by MTT assay
or fixation with 4% PFA at room temperature for 30 min for further immunostaining
with anti-TH antibody.
Primary glia cells were isolated from newborn C57BL/6 mice according to the
previously described procedures.3 Briefly, ventral midbrains were dissected, freed
from meninges, minced and digested with 0.25% trypsin and 0.01% DNase. The cells
were harvested by centrifugation, resuspended in the complete DF12 medium
supplemented with 10% FBS and seeded in T75 flasks at a density of 2×107 cells per
flask. After culture for 2 weeks, the flasks were shaken at 190 rpm for 18 hrs at 37°C
to separate microglia and astrocytes. The floating cells were then collected as
microglia cells and cultured for treatment. The adherent cells, most of which were
astrocytes, were allowed to grow for another 3 days. And the flasks were shaken again
to remove the remaining microglia cells to finally obtain the astrocytes.
Immunoblotting
Total proteins were isolated from homogenized ventral midbrain tissues or
cultured cells with RIPA lysis buffer (Beyotime). Aliquots of proteins were separated
on a SDS-PAGE gel and transferred onto PVDF membranes. The nonspecific sites
were blocked in 5% nonfat milk or 5% BSA for 1 hour at room temperature. The
membranes were then incubated overnight in the presence of the primary antibodies
against the following proteins: iNOS (1:1 000; Cell Signaling, Danvers, MA, USA),
p-NFκB (1:1 000; Cell Signaling), TH (1:1 000; Millipore, Billerica, MA, USA),
IL-1β (1:1 000; Biovision, Milpitas, CA, USA), Jmjd3 (1:200; Sigma), UTX (1:1 000;
Abcam, UK), H3K27me3 (1:1 000; Cell Signaling), H3 (1:2 000; Cell Signaling),
cleaved-caspase 3 (1:1 000; Cell Signaling) and PARP (1:1 000; Cell Signaling). A
mouse anti-β-actin antibody (1:30 000; Sigma) was used to demonstrate equal protein
loading. After that, the membranes were washed with PBS and incubated with
secondary antibodies conjugated with horseradish peroxidase (HRP) for 2 hrs at room
temperature. Proteins were finally detected using the SuperSignal Detection Kit
(Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
Immunostaining
To perform immunofluorescence, the fixed primary neurons or brain sections
were incubated in blocking buffer (PBS containing 5% serum and 0.3% Triton-X-100)
at room temperature for 30 min, followed by an overnight incubation at 4°C with the
primary antibodies against the following proteins: Iba1 (1:1 000; Wako, Japan), TH
(1:800; Millipore) and H3K27me3 (1:1 000; CST). In some cases, the sections were
double stained with anti-TH (1:200, Sigma) and anti-Iba1 (1:1 000; Wako) antibodies.
The sections were then washed with PBS and incubated with secondary antibodies
conjugated with Alexa Fluor-488/555 (1:400; Invitrogen) at room temperature for 2
hrs. The sections were then washed with PBS and the nuclei was stained with Hoechst
33342. Finally, the sections were visualized under a fluorescence microscopy
(Olympus IX81).
Immunohistochemical staining was performed using the VECTASTAIN ABC
kit (Vector Labs, Burlingame, USA). Briefly, fixed neurons or brain sections were
treated with 0.3% H2O2 for 12 min and then blocked at room temperature for 30 min,
followed by incubation overnight at 4°C with anti-TH antibody (1:800; Millipore).
The neurons or sections were then washed and incubated with biotinylated secondary
antibody (1:200; Jackson ImmunoResearch, Bar Harbor, USA) at room temperature
for 2 hrs. After that, the neurons or sections were incubated with the ABC reagent
(Vector Labs) for 45 min and stained with the diaminobenzydine (DAB) solution
(Vector Labs). Before viewing under the microscopy, the sections were dehydrated in
an ascending ethanol series and then cleared in xylene followed by mounting with the
neural resin.
ChIP assay
ChIP assay was carried out using Pierce Agarose ChIP Kit according to the
manufacturer’s instructions. Briefly, ~2×106 N9 cells were crosslinked in 1%
formaldehyde, quenched with glycine, washed with ice cold PBS and harvested into
tubes by scraping. Cell pellets were lysed in Lysis buffer containing proteinase
inhibitors and the nuclei was then digested with Micrococcal Nuclease. The yielding
sheared chromatin was immunoprecipitated with 4 μg anti-H3K27me3 (Millipore)
and 3.5 μg anti-Jmjd3 (Abcam) antibodies followed by incubation with A/G plus
agarose resin. Resin were then washed and binding DNA were recovered after 65°C
incubation. Purified ChIP DNA were measured by semi-quantitative PCR using
primers specific to Arginase1 promoter (Forward primer: AGT CAG AGA GCA GAA
GGC, and reverse primer: ACC CAA AGT GGC ACA AC). Data are presented as
enrichment of ChIP DNA levels normalized to input DNA levels.
Measurement of arginase activity
Arginase activity was measured using QuantiChrom Arginase Assay Kit
(Bioassay systems, Hayward, CA, USA) according to the manufacturer’s instructions.
Briefly, N9 cells were digested and centrifuged at 1 000g at 4°C for 10 min. Lyse cell
pellets for 10 min in 100 µl of 10 mM Tris-HCl (PH 7.4) containing 0.4% Triton
X-100. Centrifuge lysates at 14 000g at 4°C for 10 min. The supernatant was mixed
with substrate buffer and reacted with L-arginine at 37°C for 2 hrs to produce urea,
which was then incubated with urea reagent. OD520nm was recorded and calculated
into arginase activity. One unit of arginase converts 1 µmole of L-arginine to ornithine
and urea per minute.
References
1
Kingston RE, Chen CA, Rose JK. Calcium phosphate transfection. Curr
Protoc Mol Biol 2003; Chapter 9: Unit 9.
2
Studer L. Culture of substantia nigra neurons. Curr Protoc Neurosci 2001;
Chapter 3: Unit 3.
3
Yang D, Peng C, Li X, et al. Pitx3-transfected astrocytes secrete brain-derived
neurotrophic factor and glial cell line-derived neurotrophic factor and protect
dopamine neurons in mesencephalon cultures. J Neurosci Res 2008; 86:
3393-3400.
Figure Legends To Supplementary Figures
Supplementary Figure S1 Microglia can be polarized into different functional
phenotypes. (A) N9 microglia were treated with LPS (100 ng/ml)/IFNγ (10 ng/ml) as
M1 triggers and IL-4 (10 ng/ml)/IL-13 (10 ng/ml) as M2 triggers respectively for 24
hrs. The mRNA levels of M1 and M2 markers were determined by real-time PCR. (B,
C) N2A neurons were cultured with the CM from treated Raw264.7 macrophages (B)
or N9 microglia (C) for 24 hrs. MTT assay was then performed to assess the neuron
viability. (D) The content of nitrate in the CM from treated N9 cells was measured
using Griess reagent. (E) The primary VM neurons were cultured with CM from N9
cells treated with M1 or M2 triggers separately. The morphology of DA neurons were
visualized by immunostaining with anti-TH antibody. Scale bar, 100 μm. *p< 0.05,
**p<0.01, ***p< 0.001.
Supplementary Figure S2 Jmjd3 knockdown impairs the polarization of M2c
microglia. N9 microglia were treated with IL-10 (10 ng/ml) for 24 hrs. The mRNA
level of Arginase1 was determined by real-time PCR. *p<0.05, ***p<0.001.
Supplementary Figure S3 The iNOS inhibitor (L-NAME) antagonizes the
Arginase1 inhibitor (nor-NOHA) in the NO production. PM cells were treated
with nor-NOHA (100 uM) and L-NAME (100 uM) for 24 hrs. CM were collected and
NO production was measured. ***p< 0.001.
Supplementary Figure S4 Extracellular arginine transportation is not affected by
Jmjd3 knockdown. N9 cells were infected with pLKO-kdNC, pLKO-kdJmjd3, as
well as pLKO-kdJmjd3/pLVX-Arginase1 mixed lentiviruses. The infected N9 cells
were pretreated with L-arginine (0.5 mM, 1 mM, 5 mM) for 4 hrs respectively before
LPS (100 ng/ml) insult for another 24 hrs. CM were then collected and NO
production was measured by Griess reagent. ***p< 0.001.
Supplementary Figure S5 Microglia activation in the MPTP injected mice.
C57BL/6 mice (male, 12 weeks old) were injected with MPTP (20 mg/kg i.p.) for 4
times at 2-hr intervals. Saline was injected as control. Mice were then sacrificed 1 day,
3 days, 7 days or 14 days after the last MPTP injection. (A-E) The brain sections were
stained with anti-Iba1 antibody to visualize microglia activation in the SN at different
time points after MPTP injection. Scale bar, 200 μm. (G-H) The brain sections were
stained with anti-Iba1 antibody to visualize microglia activation in the striatum one
day after MPTP injection. Scale bar, 200 μm. (F, I) Quantification of Iba1
immunoreactive cells in the SN (F) and striatum (I). **p<0.01, ***p< 0.001.
Supplementary Figure S6 H3K27me3 level is higher in the SN of aged mice. The
H3K27me3 level in the midbrain of young (2-3 months) and old mice (16-18 months)
was assessed by immunostaining with anti-H3K27me3 antibody. Scale bar, 200 μm.