(+++)

Normal breast

ATM

(++)IDC

ATM

Lymph node metastasis negative positive

P-value

-

+

++

+++

35

32

27

16

36

73

50

27

0.085

A

BX200 X200

C

Vector ATM - WT ATM – S1981A

CPT

Figure S1. ATM expression does not correlate with lymph node metastasis in breast cancer specimens. (A) ATM expression was detected in the 296 cases of breast cancer tissues. Representative images from an IDC sample or a normal breast tissue was shown. (B) The Chi-square test was performed to determine the correlation of ATM expression and lymph node metastasis. (C) GM9607 cells were transfected with vector, the wild-type or S1981A form of ATM followed by immunohistochemistry in the cultured cells.

Sun et al Figure S1

Kaplan-Meier Curve:

D

P=0.047

DFP: disease free period

Snail

pS1981-ATM

P=0.264

E

Sun et al Figure S1

Figure S1. (D) A picture with both tumor cells (in the left area) and a normal breast duct (arrow). The size of the breast tumor cells are much bigger than the normal epithelial cells of breast duct. Kaplan-Meier Curves correlated with Snail (E) and pS1981-ATM (F) in 296 patients were created and statistically quantified.

F

Snail

b-actin

KU - + - + - + - + CPT (hr) 0 2 0 2

ctrl shRNA ATM shRNAA

Figure S2. Snail upregulation is regulated by activation of ATM in response to CPT and IR treatment. (A) HeLa cells with proficient or deficient ATM were treated with CPT with or without Ku55933. Total cell lysates were obtained and the Snail and β-actin levels were determined. (B) MDA-MB231 cells were exposed to IR (6 Gy), and immunoblotted with Snail and actin antibodies. (C) MDA-MB-231 cells were transfected by two different ATM siRNAs, followed by CPT and IR treatment. pS1981-ATM, ATM, Snail and actin levels were examined by immunoblotting.

Sun et al Figure S2

IR (6 Gy) 0 1 hr 2 hr 4 hr

Snail

β-actin

MDA-MB-231B

MDA-MB-231

β-actin

ATM

Snail

- CPT IR - CPT IR - CPT IR

Ctrl siRNA ATM siRNA #1 ATM siRNA #2

pS1981-ATM

C

Snail

ATM

GSK3b

CPT - + + + + - + + + + KU (mM) - - 1 5 10 - - 1 5 10

MDA-231 control MDA-231 GSK3b KD

Figure S3. GSK-3β is dispensable for ATM mediated Snail upregulation. control or GSK-3β knock-down MDA-MB-231 cells were treated with Ku55933 at indicated doses, followed by CPT treatment. Snail, ATM and GSK-3β levels were measured by Western blot.

Sun et al Figure S3

pS100-Snail

mg 10 20 40 80 10 20 40 80

Non-phosphor-Snail peptide Phosphor-S100-Snail peptide

Figure S4. The specificity of anti phospho-S100 Snail antibody. Non-phospho or phospho-S100 Snail peptides were blotted using the anti-phospho S100-Snail antibody.

Sun et al Figure S4

HSP90 alpha

HSP90 beta

Figure S5. Mass-spectrometry data for HSP90 alpha and beta binding to Snail in response to CPT treatment. MDA-MB-231 cells were treated with CPT, followed by immunoprecipitation of Snail, electrophoresis, silver staining and mass-spectrometry. The Spectra of HSP90 alpha and beta were shown.

Sun et al Figure S5

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

1* 2 3 4* 5 6* 7 8 9* 10

* Breast cancer with lymph node metastasis

11* 12 13 14 15* 16 17 18 19 20

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

21* 22 23* 24 25 26* 27 28 29* 30

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

Sun et al Figure S6

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

31* 32 33 34 35* 36 37* 38* 39* 40

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

41 42* 43* 44 45 46 47 48 49 50

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

51 52 53 54* 55 56 57* 58 59* 60

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

61* 62 63* 64 65 66 67* 68 69 70

-actin

pS100-Snail

Snail

pS1981-ATM

ATM

71 72 73* 74 75 76* 77 78 79 80*

Figure S6. Snail, pS100-Snail, ATM, pS1981-ATM and -actin were examined by immunoblotting in 80 breast cancer patient tissue samples.