Cell - 8 September 2016

*

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).

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