Gene Expressio n. (fold change. ) ... B5E2 PDK4 BDNF CYCS Cox5b. 0.0. 0.5. 1.0 ... (D) GSK3β protein and gene expression 48 hours following GSK3β siRNA.
Cell Reports, Volume 23
Supplemental Information
GSK3b Regulates Brain Energy Metabolism Stephen A. Martin, Dylan C. Souder, Karl N. Miller, Josef P. Clark, Abdul Kader Sagar, Kevin W. Eliceiri, Luigi Puglielli, T. Mark Beasley, and Rozalyn M. Anderson
4.0 nmol/mg
pO 2
Control siGSK3β
3.0 2.0
pGSK-3β/GSK-3β
0
60
Minutes
50
40
30
10
20
0
*
1.0
0
Relative Protein
NADt
UT 24h LiCl
Con GSK3β S9A
Tomm20
1.5
Gene Expression (fold change)
Minutes
UT
1.5
LiCl
1.0
1.0
0.5
0.5
0.0
0.0
CI
CII
CIII
CIV
PGC-1α
CV
DAPI
2.0
UT
LiCl + LiCl VIII VIII
90 kD 70 kD
0.5
*
1.5
*
Con
1.0 0.5
siGSK3β
0.0
UT 2h 24h
UT LiCl VIII
LiCl
pGSK3β
DAPI
Merged
LiCl + VIII
P Gene Expression (fold change)
UT
0
K
24h
PGC-1α
24h LiCl
0
N
* *
Tubulin
50 25
M
1.0
O
UT LiCl VIII LiCl+VIII
*
UT 24h LiCl
Relative Protein
25
0.0
0.5
*
*
*
75
J Stain Intensity (fold change)
100
Con siGSK3β
Relative Protein
I
50
1.5
* 1.0
0.0
75
L
100
Ponceau
n β Co SK3 siG 24h VIII
*
60
HA
n β Co SK3 siG
0.5
50
_
GSK3b
1.0
G
S9A
40
LiCl
*
*
24h LiCl 1.5
pcDNA3.1
2.0
0.0
24h 24h VIII LiCl + VIII
30
24h 24h UT LiCl
1.0
0.0
F
3.0
0.0
0
Ponceau
20
24h LiCl + VIII
0.5
siGSK3β
10
24h VIII
GSK3β
Control
pO 2
24h LiCl
ND ND
+
Membrane Potential
24h UT
2
1.0
Gene Expression (fold change)
Day 0
4
LiCl
UT 1m M 2. 5m M 5m M 15 m M
0
H
Membrane Potential
Relative Cell Death %
100
6
_
Relative Cell %
+
*
1.5
+
200
8
1.5
* * *
2.0
_
*
D
*
+
10
300
E
C
_
B
* vs. 24h UT + vs. 24h UT and 24h VIII
+
A
Con
2.5
*
2.0
siGSK3β
1.5 1.0 0.5 0.0
Pan
α1
α4
B5E2 PDK4 BDNF CYCS Cox5b
PGC-1α
Figure S1. (related to Fig. 1). Data show the impact of lithium on cell proliferation (A) and (B) cell death. (C) JC-1 measurement of mitochondrial membrane potential following LiCl dose-response in H4 glioma. (D) GSK3β protein and gene expression 48 hours following GSK3β siRNA transfection. (E) JC-1 measurement of mitochondrial membrane potential following GSK3b inhibitor VIII (15μM) in H4 glioma with GSK3β interference. (F) GSK3β protein and gene expression 48 hours following GSK3β−S9A transfection. (G) Basal oxygen consumption over time in H4 glioma treated with DMSO, LiCl (15mM), inhibitor VIII (15μM), and both LiCl (15mM) and inhibitor VIII (15μM), and (H) in H4 glioma with GSK3β interference. (I) Total NAD (NADt) levels, and immunodetection of Tomm 20 (J), complexes I, II, III, IV, and V proteins of the ETS (K), and pGSK3β/GSK3β ratio (L) following the indicated LiCl treatment (15mM) in H4 glioma. (M) Detection of PGC-1α protein in H4 glioma following the indicated treatment. (N) Immunodetection of PGC-1μM in H4 glioma with GSK3β interference. (O) Immunodetection of tubulin and pGSK3β following 24h LiCl treatment in H4 glioma. (P) Gene expression of PGC-1μM and indicated transcripts in H4 glioma with GSK3β interference. (n= 3-6 biological replicates per assay; data shown as average +/- SEM; *p
