Supplemental Figures
Figure S1. Dyn/KOR in co-cultured microglia protects TH
+ neurons from LPSinduced apoptosis and pathological morphology
(a) Schematic of microglia/neuron co-culture procedure.
(b) TH
+
cell number of neurons directly treated with LPS for 2 h (neuron), or co-cultured
24 h with microglia that had been LPS-treated for 2 h (co-culture).
(c) Morphological changes of TH + neurons co-cultured for 24 h with WT or Kor -/microglia (Ctrl) or with LPS- or LPS+Dyn-stimulated microglia (LPS, LPS+Dyn).
TH
+ neurons with decreased dendrite numbers were regarded as pathological TH+ cells.
(d) Immunofluorescence staining of neurons with pSIVA and PI, indicating apoptotic cells.
Values are the mean±SD in separate experiments
(n=3 ) with significance
* p <0.05,
** p <0.01 vs respective control (no treatment) group (Student’s t -test).
1

Figure S2. Multiple KOR agonists inhibit, and KOR knockdown restores, LPSinduced neurotoxic factor production and pro-inflammatory signaling in microglia
(a), Suppression of IL-1ß, TNF-

and iNOS mRNA level by different KOR agonists in murine microglial BV2 cells activated by LPS co-administration.
(b) Effects of Dyn co-administration on 2 h LPS-induced TNF-

production in KOR siRNA-knockdown vector transfected (Si kor ) murine microglial BV2 cells.
(c) Effects of Dyn co-administration on 2 h LPS-induced IKK phosphorylation and LPSinduced IkB degradation in KOR knockdown (Si kor ) BV2 cells.
Values are the mean±SD in separate experiments (n=3 ) with significance
* p <0.05,
** p <0.01 vs respective control (no treatment) group (Student’s t -test).
2

Figure S3.
KOR activation blocks microglial NF-kB p65 nuclear translocation
(a) NF-kB p65 fluorescence image of WT or Kor
-/microglia treated for 2 h with LPS and/or Dyn. Right panel indicates quantification of NF-kB p65 fluorescence intensity in the cytoplasm (Cyt) and nucleus (Nuc).
(b) Expression of NF-kB p65 in siRNA control (SiCtrl) and KOR siRNA (Si kor ) transfected murine microglial BV2 cells after 2 h treatment with LPS and/or Dyn.
(c) Effects of 2 h KOR agonist and/or antagonist (Dyn and/or NorB) treatment on LPSinduced and uninduced NF-kB p65 expression in Nuc and Cyt of BV2 cells.
Values are the mean±SD in separate experiments (n=3 ) with significance
* p <0.05,
** p <0.01 vs respective control group (Student’s t -test).
3

Figure S4. Expression level of
ß
-arrestin2 mRNA in BV2 cells with Dyn- administration for different duration
BV2 cells were treated with Dyn(5µM) and the mRNA level of ß-arrestin2 was detected by qPCR assay. Values are the mean±SD in separate experiments (n=3), no significant difference was detected in both Dyn treated cells comparing with control (0 min) group using Student’s t-test.
Figure S5. Dyn suppresses LPS-induced microglial activation
LPS or LPS+Dyn were injected into WT mice’s left substantia nigra (SN). The animals were sacrificed after 24 h for immunohistological study. To detect activation of microglia, IHC was performed using the Vector ® Elite ABC kit (Vector Laboratories), following overnight incubation of brain sections with primary antibody (rabbit anti-
CD11b, 1:500, Santa Cruz). Then the sections were developed with DAB and mounted for microscopic analysis (Zeiss Axioplan 2).
(a) CD11b staining of microglia in mice brain (with magnification of inset SN area, revealing density of stained microglia, at right).
(b) Quantification of number of stained microglia from panel (a) in SN 24 h after LPS injection.
Values are the mean±SD in separate experiments
(n=3 ) with significance
* p <0.05,
** p <0.01 vs respective control (uninjected) group (Student’s t -test).
4

1
2
3
4
5
Figure S6. Counting of TH + -neuron number
After the cells were labeled with TH (Red), NeuN (Green) and DAPI (Blue), a counting frame was superimposed on the stack of 40× images. The right boundary and the upper boundary of the counting frame were used as acceptance lines; the other two boundaries were forbidden lines. The TH-positive neurons were counted only if NeuN-labeled nuclei and TH-immunoreactive profiles fell entirely inside the counting frame or if they crossed an acceptance line without also crossing a forbidden line. NeuN-labeled neurons were counted when the top of the nucleus was found within the thickness of the tissue section.
Table S1: The TH + - neuronal counts from all animal groups.
wt Kor -/-
Animals
Saline LPS LPS+Dyn Saline LPS LPS+Dyn
6353
6643
7865
7234
6452
4636
3873
5024
2653
2722
4827
5737
4624
4642
6422
6535
7533
6474
6422
6962
2136
2742
1744
3421
2424
2644
1648
2452
3253
3522
5