Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Shihao Du1, 2, 5, Xiaoyu Song3, 5, Yuan Li2, 5, Yalei Cao1,5, Fuhao Chu1, Olanrewaju Ayodeji Durojaye3, Zeqi Su1, Xiaoguang Shi2, Jing Wang2, Juan Cheng2, Tangshun Wang2, Xiang Gao2, Yan Chen1,2, Wuzhekai Zeng3, Fengsong Wang4, DongMei Wang3, Xing Liu3, Xia Ding1,2,*

1 School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China 100029; 2 Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China 100700; 3 MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics & Hefei National Center for Physical Sciences at the Microscale, University of Science & Technology of China, Hefei, China 230027; 4 School of Life Science, Anhui Medical University, Hefei, China 230032.

5 Equal contribution

*Correspondence: dingx@bucm.edu.cn

Supplemental Figure 1

B

A

Figure S1A. The inhibitory effect of several TCM monomers (1μM) on MHCC97H cell motility were tested by wound

healing assay. The wound healing ratio (%) of 8 hours were calculated. B. MHCC97H cells were treated with 2‰ DMSO and different concentration of celastrol (5 nM, 10 nM, 50

nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM) for 24h before MTS assay, the cell viability was calculated by dividing the OD of each group by OD of the control group.

C. HepG2 cell were treated in the same way as above. The error bars represent S.E.; n = 3 preparations, *P<0.05, **P < 0.01.

C

60

100

20

60

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20 Cel

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y (%

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ell v

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(%)

10 100 1000

Celastrol, nM10000

60

100

20

10 100 1000

Celastrol, nM10000

Du et al., W

ound

hea

ling

(%)

Supplemental Figure 2

B

A

Figure S2A. Wound healing area of Huh7 cell. Images were collected before and 8 hours after 20% fetal calf

serum addition. 2‰ DMSO were added to the Control group and 500 nM celastrol were added to the treatment group. Bar = 100 μm.

B. Wound healing area of HepG2 cell.

0 h 8 h

DM

SOC

elas

trol

Huh7

HepG2

DM

SOC

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trol

0 h 8 h

Du et al.,

Supplemental Figure 3

B

A

Figure S3A. The flowchart shows the synthesis process of celastrol-biotin. B. The mass spectrometry identified the celastrol-biotin (peak 719.4204).

Celastrol-biotin

HOBT/EDCI/DIPEADry DMF

0.6

0.2

1.0

×106

Counts vs. Mass-to-Charge (m/z)

Du et al.,

Supplemental Figure 4

BA

Figure S4A. The coomassie brilliant blue (CCB) stain result shows the protein pulled down by celastrol-biotin.

The MHCC97H cell lysates were treated with 10 μM celastrol-biotin and equal volume of DMSO (-), and then incubated with avidin-beads.

B. Western-blotting result of the co-immunoprecipitation assays. HEK293T cells transfected with Flag-ROCK2 and GFP-ezrin were lysed and incubated with FLAG M2 beads, followed by the western blotting with anti-GFP and anti-FLAG antibodies. The transfected cells were treated with 500 nM celastrol for 6 hours, while the control group were treat with equal volume of DMSO. The cells transfected with Flag-ROCK2 and GFP vector were negative control. Full-length blots are presented in Supplementary Figure S8.

CBBStaining

Mr(K)

1 2

+ - Celastrol-Biotin

Immuno-Precipitation:

FLAG Antibody

1 2 3 4 5 6

GFP blotting

GFP-ezrin

IgG (HC)

FLAG-ROCK2

Input IP

GFP

GFP-ezrin +CelastrolGFP-ezrin

FLAG-ROCK2

＋ － － ＋ － － － ＋ － － ＋ － － － ＋ － － ＋ ＋ ＋ ＋ ＋ ＋ ＋

Input:GFP-ezrin

Mr(K)

116-97-

66-

44-

29-

Du et al.,

130-180-

70-

55-

40-

35-

100-

25-

200-

Residues Distance (A0)

MET-172ASP-218ASP-232

2.45.62.8

A

B

Supplemental Figure 5

Figure S5A. Cartoon representation of Y27632 (green) docked onto the ROCK2 model. Potential area of Y27632

interacted with the residues of ROCK2 were shown in yellow dotted lines. The numbers represent the distance between each residue and Y27632.

B. The amino acid residues of ROCK2 that formed polar bonds with Y27632 and the distance between each residue and Y27632.

Du et al.,

A

B

Supplemental Figure 6

Figure S6A. Western blotting analyses of ezrin pT567, ezrin and Tubulin, after treating Huh7 cells with different

concentration of celastrol for 6h. Full-length blots are presented in Supplementary Figure S8.B. Western blotting analyses of ezrin pT567, ezrin and Tubulin, after treating HepG2 cells with different

concentration of celastrol for 6h. Full-length blots are presented in Supplementary Figure S8.

Du et al.,

Mr(K)

ezrin

Tubulin

Celastrol, nM 0 5 50 500 5000Mr(K)

1 2 3 4 5

80- ezrin pT567

80-

50-

ezrin

Tubulin

Celastrol, nM 0 5 50 500 5000

1 2 3 4 5

80- ezrin pT567

50-

80-

Supplemental Figure 7

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Du et al.,

Supplemental Figure 8Figure 2 B Figure 3 A

80- ezrin

Du et al.,

50-

Figure 4 E

180-

Mr(K)

180- ROCK2

50- Tubulin

80-

Mr(K)

ezrin pT567

Figure S6B

ezrin pT567Mr(K)

Mr(K)

50-

Tubulin

Figure S4B

116-

200-

66-

29-

97-

44-

Mr(K)FLAG-ROCK2

50- Tubulin

ezrin pT567Mr(K)

80-

Figure 3 B

130-

180-

70-

100-

80-

80-

ezrin

Figure 2 E

50-

180-

Tubulin

Mr(K)

Flag-ROCK2

Figure S6A

Tubulin

ezrin pT56780-

80- ezrin

** non-specialMr(K)

80- ezrin

Figure 4 A

Mr(K)

80-

200-

116-97-

66-

44-

29-

ezrin pT567

Flag-ROCK2

His-ezrin

Figure 4 B

Mr(K)

80-ezrin pT567

29-

44-

66-

97-116-

200- Flag-ROCK2

His-ezrin

Tubulin

ROCK2 pS1366

50- Tubulin

ROCK2Mr(K)