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A ATM (+++) (++) X200 X200 B IDC Normal breast C 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. ATM – S1981A Vector ATM - WT Sun et al Figure S1
D Snail E Kaplan-Meier Curve: P=0.047 F pS1981-ATM P=0.264 DFP: disease free period 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 muchbigger 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. Sun et al Figure S1
MDA-MB-231 pS1981-ATM ATM Snail β-actin A B Snail MDA-MB-231 β-actin IR (6 Gy) 0 1 hr 2 hr 4 hr ctrl shRNA ATM shRNA Snail C b-actin KU - + - + - + - + CPT (hr) 0 2 0 2 - CPT IR - CPT IR - CPT IR Ctrl siRNA ATM siRNA #1 ATM siRNA #2 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
Snail ATM 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. GSK3b CPT - + + + + - + + + + KU (mM) - - 1 5 10 - - 1 5 10 MDA-231 control MDA-231 GSK3b KD 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
1* 2 3 4* 5 6* 7 8 9* 10 Snail Snail Snail pS100-Snail pS100-Snail pS100-Snail ATM ATM ATM pS1981-ATM pS1981-ATM pS1981-ATM b-actin b-actin b-actin 31* 32 33 34 35* 36 37* 38* 39* 40 Snail pS100-Snail 11* 12 13 14 15* 16 17 18 19 20 ATM pS1981-ATM b-actin 51 52 53 54* 55 56 57* 58 59* 60 41 42* 43* 44 45 46 47 48 49 50 Snail Snail pS100-Snail pS100-Snail ATM ATM 21* 22 23* 24 25 26* 27 28 29* 30 pS1981-ATM pS1981-ATM b-actin b-actin 61* 62 63* 64 65 66 67* 68 69 70 71 72 73* 74 75 76* 77 78 79 80* Snail Snail pS100-Snail pS100-Snail ATM ATM pS1981-ATM pS1981-ATM b-actin b-actin *Breast cancer with lymph node metastasis Figure S6. Snail, pS100-Snail, ATM, pS1981-ATM and b-actin were examined by immunoblotting in 80 breast cancer patient tissue samples. Sun et al Figure S6
