Supplementary Materials for

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1 Supplementary Materials for The tyrosine phosphatase (Pez) inhibits metastasis by altering protein trafficking Leila Belle, Naveid Ali, Ana Lonic, Xiaochun Li, James L. Paltridge, Suraya Roslan, David Herrmann, James R. W. Conway, Freya K. Gehling, Andrew G. Bert, Lesley A. Crocker, Anna Tsykin, Gelareh Farshid, Gregory J. Goodall, Paul Timpson, Roger J. Daly, Yeesim Khew-Goodall* The PDF file includes: *Corresponding author. Published 17 February 2015, Sci. Signal. 8, ra18 (2015) DOI: /scisignal Fig. S1. knockdown promotes invasiveness in MDA-MB-231 cells. Fig. S2. promotes the secretion of TGFβ in human breast cancer cell lines. Fig. S3. Relative cytokine abundance in conditioned media from breast cancer cells after overexpression or knockdown. Fig. S4. Relative abundance of cytokine mrna transcripts in breast cancer cells after overexpression or knockdown. Fig. S5. Schematic of the injection protocol used to test the effect of conditioned medium on the growth and metastasis of breast cancer cells in mice. Fig. S6. Identification of substrates by differential phosphoproteomics. Fig. S7. Total protein abundance of candidate substrates is unaffected by knockdown. Fig. S8. Western blot analyses to ascertain overexpression or knockdown efficiencies. Fig. S9. Correlation between technical replicate SILAC-MS experiments. Table S1. List of candidate (Pez) substrates from SILAC-MS screen. References (53 62)

2 Supplementary Materials. A B 231- Invasion Proliferation 231-sh Number of invaded cells *** sh Proliferative index [%] Cells within matrix sh Proliferative index [%] Cells on top of matrix *** sh Figure S1: knockdown promotes invasiveness in MDA-MB-231 cells. The invasive capacity of control MDA-MB-231 cells (231-) and knockdown cells (231-sh) was assessed by 3-D organotypic collagen I invasion assays. (A) Representative images of H&E stained sections from invasion assays after 12 days of invasion. Scale bars, 100 µm. Quantification of invasion (A), and proliferation (B) as measured by Ki67 staining of cells within or on top of the matrix. Data are means ± SEM from 3 independent experiments. **p<0.01, Student s t-test.

3 MDA-MB-468 MDA-MB-231 TGF-beta1 (pg/ml) TGF-beta1 (pg/ml) Figure S2: promotes the secretion of TGFβ in human breast cancer cell lines. Total TGFβ1 abundance in conditioned media from 468-EV, 468- (n=3 clones each), 231- and 231- sh clones (n=4 clones each) measured using a TGFβ1 ELISA. *p<0.05, two-tailed t-test.

4 Figure S3: Relative cytokine abundance in conditioned media from breast cancer cells after overexpression or knockdown. Quantitation of raw data (total pixels) from chemiluminenscent detection of array membranes for cytokine content in MDA-MB-468 CM pooled from 3 clones (top panel) and MDA-MB-231 CM pooled from 4 clones (bottom panel). Each symbol represents a duplicate spot on the array.

5 Relative mrna MDA-MB-468 p=0.02 * EV 0 6 MDA-MB-231 sh Relative mrna 4 2 p=0.009 ** p=0.01 * 0 Figure S4: Relative abundance of cytokine mrna transcripts in breast cancer cells after overexpression or knockdown. The abundance of mrna transcripts that encode cytokines displaying altered presence in conditioned medium upon overexpression or knockdown was quantified in 468-EV (n=3 clones), 468- (n=3 clones), 231-NC (n=4 clones) and 231-sh (n=4 clones) by quantitative RT-PCR, and normalized to β-actin internal control. P values were derived using a twotailed t-test.

6 Daily IP injections of CM for 3 days (pre-conditioning) Implant cells into MFP of both sets of mice cells Continued daily IP injections of CM for 30 days sh CM CM sh Figure S5: Schematic of the injection protocol used to test the effect of conditioned medium on the growth and metastasis of breast cancer cells in mice. MFP, mammary fat pads; IP, intraperitoneal; CM, conditioned medium;, control shrna-transfected cell cultures; sh, - targeted shrna-transfected cell cultures.

7 A actin B sh cells cells Figure S6. Identification of substrates by differential phosphoproteomics. (A) Representative Western blotting analysis for and Actin in MDA-MB-231 cells stably expressing a shrna (sh) or non-targeting shrna (). (B) Workflow for SILAC-MS phosphoproteomics to identify proteins that are differentially tyrosine phosphorylated when is reduced.

8 INPPL1 IRS1 actin RIN1 EEF1A1 actin TYK2 actin Figure S7: Total protein abundance of candidate substrates is unaffected by knockdown. Representative Western blotting analysis of 231- and 231-sh cell lysates for a selection of candidate substrates identified in the SILAC-MS screen.

9 A RIN1 actin GFP IRS1 B RIN1 PRKCD C D RIN1 RIN1 Figure S8: Western blot analyses to ascertain overexpression or knockdown efficiencies. (A) Western blotting in lysates from MDA-MB-468 cells transfected with the indicated plasmids. Unlabeled lane is an irrelevant sample. (B) Western blot analysis of lysates from primary human lymphatic endothelial cells transfected with the indicated sirnas. (C and D) Western blotting analysis of lysates from transfected BT549 cells. Blots are representative of 3 experiments.

10 Experiment 1 sh(h):(l) Technical Replicates A/B Experiment 2 sh(l):(h) (Inverse) Technical Replicates A/B A A B A B B SILAC Ratio (B) SILAC Ratio (B) PY100 IP SILAC Ratio SILAC Ratio (A) C A B A B D SILAC Ratio (B) SILAC Ratio (B) PY20 IP SILAC Ratio SILAC Ratio (A) E SILAC Ratios (Exp 2) SILAC Ratios (Exp

11 Figure S9. Correlation between technical replicate SILAC-MS experiments. Venn diagrams indicating degree of overlap for high confidence phospho-peptides from technical replicates from Experiments 1 and 2 for py100 (A) and py20 IPs (C). Correlation coefficients (R2) for phosphotyrosine peptide SILAC ratios from technical replicates from Experiments 1 and 2 for py100 (B) and py20 IPs (D). (E) Correlation between biological replicate SILAC-MS experiments for peptides displaying at least +/-1.2- fold change in both experiments. The correlation coefficient (R2) and p values were obtained by linear regression analysis (GraphPad Prism).

12 Candidate UniProt Modified Fold change Name Substrate ID site Exp 1 Exp 2 Role of phosphorylation at identified tyrosine residue >1.2-fold (Exp 1&2) MK01 P28482 Mitogen-activated protein kinase 1 (ERK2) Tyr Activation of kinase activity 53 DYRK1A Q13627 Dual specificity tyrosine-phosphorylation-regulated kinase 1A Tyr Non-nuclear localisation 54 RIN1 Q13671 Ras and Rab interactor 1 Tyr Abl kinase activation / EGFR stabilisation 55 MK03 P27361 Mitogen-activated protein kinase 3 (ERK1) Tyr Activation of kinase activity 53 EF1A1 P68104 Elongation factor 1-alpha 1 Tyr Unknown P85B O00459 Phosphatidylinositol 3-kinase regulatory subunit beta Tyr Unknown PRP4B Q13523 Serine/threonine-protein kinase PRP4 homolog Tyr Unknown >1.2-fold (Exp 1) KPCD Q05655 Protein kinase C delta type Tyr <1.2 Unknown ACK1 Q07912 Activated CDC42 kinase 1 Tyr <1.2 Associated with clathrin-coated pit formation 56 CALM P62158 Calmodulin Tyr <1.2 Enhanced interaction with binding partners 57 SHB Q15464 SH2 domain-containing adapter protein B Tyr <1.2 Unknown SHB Q15464 SH2 domain-containing adapter protein B Tyr <1.2 Unknown TYK2 P29597 Non-receptor tyrosine-protein kinase TYK2 Tyr <1.2 Unknown DYRK1A Q13627 Dual specificity tyrosine-phosphorylation-regulated kinase 1A Tyr <1.2 Activation of kinase activity 58

13 GSK3A P49840 Glycogen synthase kinase-3 alpha Tyr <1.2 Activation of kinase activity 59 >1.2-fold (Exp 2) SHIP2 O15357 Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 2 Tyr 986 < Activation of phosphatase activity / ubiquitination 60,61 IRS1 P35568 Insulin receptor substrate 1 Tyr 662 < Interaction with p85 subunit of PI3K 62 Table S1: List of candidate (Pez) substrates from the SILAC-MS screen. List of proteins and tyrosine residues for which an increase in tyrosine phosphorylation was detected in cells hypomorphic for Pez in one or both replicate experiments, implicating them as candidate Pez substrates. Reported roles for tyrosine phosphorylation at these specific residues are also indicated.