*
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
DMSO PHX
Triglyceride (mM)
B
cyt. HSP70
Tubulin
Control #1 #2
mtHSP70 siRNA mtHSP70
mtHSP70
cyt. HSP70
Tubulin
DMSO 400
PHX (nM)
A
Tubulin
Relative band intensity
cyt. HSP70
mtHSP70 siRNA
0
1
2
3
4
5
6
Control #1 #2
mtHSP70 siRNA
0
0.2
0.4
0.6
0.8
1
1.2
Control #1 #2
Relative band intensity
cyt. HSP70
PHX (nM)
0
0.5
1
1.5
2
2.5
3
DMSO 400
. ***
**
*
0
1
2
3
4
5
6
mRNA Fold Induction after hspa9 knockdown (Normalized to Control siRNA-treated sample) cpt1a scd1 lpgat1 hsd17b12 acc1 acc2 srebp1 fas hspa9
*
*
*
* *
*
C
GFP
Q78
PolyQ
expressing cells
Filter Trap (anti-GFP)
mtHSP70 siRNA #2
double siRNA transfection
ACC FAS Ctrl
only
siRNA:
#1 #2 #1 #2 Ctrl
1 2 3 4 5 6
Total Protein (anti-GFP)
1 2 3 4 5 6
GFP
Q78
Tubulin
Tubulin
GFP Q78
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 1 2 3 4 5
acc1
mRNA Fold Induction after siRNA treatment(Normlized to Control siRNA-treated sample)
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 1 2 3 4 5
fas
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5 1 2 3 4 5
hspa9
**
* * * **
*
** **
**
**
**
****
****
**
*
*
*
D
.
Figure S6. MCSR Increased Expression of Cytosolic HSP70, and Fat Synthesis Is Also Required for MCSR Induction in Human Primary Fi-
broblasts, Related toFigure 7
(A) Western blotting analysis of mtHSP70 and cytosolic HSP70 (cyt. HSP70) proteins level after siRNA treatment or PHX treatment on human primary fibroblasts
(GFP expressing cells). Three independent biological repeats are shown in one gel. Relative band intensity was analyzed using ImageJ software (Bar graphs, *p%
0.05, **p%0.001,dp = 0.0560.).
(B) Triglyceride content of human primary fibroblasts (GFP expressing cells) was measured after PHX treatment (paired t test, mean±SD of three biological
repeats, p%0.05).
(legend continued on next page)