Supplementary Material

Supplemental Fig. 1. Celastrol restored the cardiac function in a DOX induced cardiomyopathy model. The DOX group showed a reduction in stroke volume to 26.14±3.0 ml/min (n=17) as compared to CEL+DOX group which showed an improvement to 47.15±6.0 ml/min (n=19, *P=0.0041). A decrease in heart rate was observed in DOX group (416±25, n= 13) as compared to CNT (503±25), but in CEL+DOX group (497±13, n=17, *P=0.041) demonstrated near normal heart rates. Similarly, cardiac ouput was preserved in CEL+DOX (24±3, n=17) as compared to DOX group (12±2, n= 17, *P=0.0007). A decrease in LVEDD after DOX treatment (2.9±0.2, n= 15) was restored back to the control levels in CEL+DOX group (3.2±0.2, n= 19). Data is represented as mean±S.E.M. Data was analyzed by one-way ANOVA followed by Kruskal-Wallis test.

Supplemental Figure 1

Supplemental Fig. 2. Quantitative analysis of mRNA expression of MMP2 in the border zone of LV infarct at 3 days post-MI (*P<0.01). Data represented as mean±S.E.M and analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Supplemental Figure 2

Control/Vehicle0.2 ml/day

CEL (4mg/kg)/day

Heart

Lungs

Intestine

Kidney

Pancreas

Spleen

CEL (8mg/kg)/day

Liver

Supplemental Fig. 3. Absence of change in tissue architecture in mice exposed to different increasing doses of Celastrol. Representative microscopic images of hematoxylin & eosin staining of the tissues harvested from CNT group (n=3), CEL4 group (4mg/kg, n=3) and CEL8 group (8mg/kg, n=3) after administrating CEL for 7 consecutive days

Supplemental Figure 3

A

Supplemental Fig. 4. Effect of celastrol treatment on cell viability. H9c2 cells were cultured in the presence of 1μM DOX (A), with or without pretreatment of celastrol as indicated. Celastrol was used at 100nM concentration for H9c2. Bar graph shows the maximum cell viability was obtained after a 24 hours pretreatment with Celastrol. *P<0.05, data is shown mean ±S.D. of three independent experiments. B). Killing curve of H9c2 cells with increasing concentration of DOX in the presence or absence of CEL.

Control 0.25μM 1μM 5μM 10μM0%

10%20%30%40%50%60%70%80%90%

100%

H9C2 Dox dose response

DoxCel 100nM + Dox

Cell

Viab

ility

B

H9C2 CEL duration response

Supplemental Figure 4.

CNT

CEL CEL+DOX

DOX

A

B

Supplemental Fig. 5. FACS analysis of Annexin V in H9c2 cells. A). Dot plot analysis of Annexin V/PI staining demonstrated that DOX treatment increases the total cell death (R1+R2) and the cells in early apoptotic stage (R2) (n=3). CEL treatment of DOX treated cells significantly reduced the cell death.CEL shows no significant effect on the cells. B). Quantitative analysis of Annexin expression. *P<0.05

R1 R1

R1 R1

R2 R2

R2 R2

Supplemental Figure 5.

Supplemental Fig. 6. Effect of DOX on mRNA expression levels of cell specific transcription factor in H9c2. DOX induced reduction in GATA-4 mRNA. Celastrol treatment restored GATA-4 to almost normal levels after CEL treatment. Results are mean ± SEM of the indicated mice. *P<0.05. Data was analyzed using one-way ANOVA, followed by Benferroni’s post hoc test.

GATA-4

Supplemental Figure 6.

Supplemental Fig. 7. Knockdown of HSF-1 in H9c2 cells. As shown in the graph, expression of Hsf-1 protein was less than 5% in HSF-1 siRNA trated samples

Supplemental Figure 7.

CEL (300nM)TAA (400nM)TTD(100mM)

Hsp70

HNF1α

HNF4α

GAPDH

_ _ _ _ _ _ _ _ _ _ _

++ +

+ + ++

+++

Supplemental Fig. 8. Immunoblot showing the effect of triptolide on expression of Hsp70 in Huh7 cells. Cells were treated with CEL, TTD, and TAA .

Supplemental Figure 8.