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1 Supplementary Materials for The lncrna H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging mir-200b/c and let-7b Wu Zhou,* Xiao-lei Ye, Jun Xu, Ming-Guo Cao, Zheng-Yu Fang, Ling-Yun Li, Guang-Hui Guan, Qiong Liu, Yue-Hui Qian, Dong Xie* *Corresponding author. (W.Z.); (D.X.) The PDF file includes: Published 13 June 2017, Sci. Signal. 10, eaak9557 (2017) DOI: /scisignal.aak9557 Fig. S1. Git2 transcript is a predicted target of mir-200b/c. Fig. S2. Cyth3 transcript is a predicted target of let-7b. Fig. S3. The functional effect of let-7 assessed with a targeted antagomir. Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays. Fig. S5. Luciferase assay results. Table S1. Metastases resulting from orthotopic injection of cells from distinct clones. Table S2. Clinical characteristics of MBC patients. Legend for data file S1 Data file S2. mirna and H19 sequences. Data file S3. mirna target information. Data file S4. ArfGEF target information. Data file S5. sirna, shrna, and PCR primer sequences. Other Supplementary Material for this manuscript includes the following: (available at Data file S1 (Microsoft Excel format). Microarray results.

2 Fig. S1. Git2 transcript is a predicted target of mir-200b/c. (A and B) Western blotting and quantification assessing the abundance of epithelial and mesenchymal markers in C13-PT, C13-PB and C13-LM cells. (C) The endogenous protein level of GIT2 in C13-PT, C13-PB and C13-LM cells was detected by Western blotting. (D) The mirna target prediction tool revealed that mouse Git2 transcript was a predicted target of mir-200b/c. (E) Conserved mir-200b/c target sites in Git2. PT, primary tumor; PB, peripheral blood; LM, lung metastasis; E-cad, E-cadherin; N-cad, N- cadherin; Vim, Vimentin. Data are means ± SD from 3 experiments; blots are representative. *P<0.05, **P<0.01, ***P<0.001, by Student s t-test.

3 Fig. S2. Cyth3 transcript is a predicted target of let-7b. (A)The H19 LncRNA of nonmetastatic TA2-C7 cells, weakly metastatic TA2-C47 cells, highly metastatic TA2-C13 cells and TA2-C13-derived sublines was analyzed by qrt-pcr, and normalized by TA2-C7 cells. (B. The Ago2 antibody precipitated the Ago2 protein from cell lysates from C13-PT, C13-PB and C13-LM cells, but the control nonimmune IgG did not. The input lane indicates lysate samples used as a positive control for Ago2. (C and D) Knockdown efficiency of three H19-targeted sirnas in TA2-C13 cells, analyzed by Western blotting (C) and quantified from RT-PCR assays (D). Ctr, Control; Si, sirna. (E and F) Targetscan results for predicted mirna binding sites in the mouse Cyth3 transcript. Red arrows and frames indicate the predicted eraction region and conserved target site sequences for let-7 mirna. Data are means ± SD from 3 experiments; blots are representative. *P<0.05, **P<0.01, ***P<0.001, by Student s t-test..

4 Fig. S3. The functional effect of let-7 assessed with a targeted antagomir. (A) let-7 mirna expression in non-metastatic TA2-C7 cells, weakly metastatic TA2-C47 cells, highly metastatic TA2-C55 and TA2-C13 cells, and TA2-C13-derived sublines was analyzed by qrt-pcr and normalized to U6 snrna. (B) Abundance of let-7 isoforms in TA2-C13 cells transfected with an antagomir of let-7, assessed with qrt- PCR. (C) Quantified results of Western blotting for CYTH3, in C13-PT, C13-PB or C13-LM cells transfected with a control or let-7 antagomir. (D) Immunohistochemistry staining for E-cadherin in the indicated cell line-derived primary tumors. Negative control is stained without E-cadherin secondary antibody. Tumors were each grown in 3 mice. (E and F) Quantified results of Western blotting for GIT2 (E) and CYTH3 (F) in the indicated cell lines or cell line-derived primary tumors ( -PT ). GAPDH served as a loading control. Data are means ± SD from 3 experiments; blots are representative. ***P<0.001; *, not significant; analyzed by Student s t-test.

5 Fig. S4. The role of GIT2 and CYTH3 as analyzed by RIP and in vitro assays. (A to D) Western blotting assessment of the effect of GIT2 knockdown (A and B) or CYTH3 knockdown (C and D) in the indicated cell lines on the amount of GTP-bound ARF6 (A, assessed by pulled down with immobilized GST-GGA3-PBD beads) and E- cadherin (B). (E and F) Western blotting assessment of the effect of mir-200b/c antagomir (E) or let-7b antagomir (F) on the abundance of E-cadherin in the indicated cells. (G and H) Western blotting assessment of the effect of H19 knockdown (with each of three targeted sirnas: #1, #2 and #3) on the abundance of E-cadherin (G) and vimentin (H) in C13-PT, C13-PB or C13-LM cells. GAPDH served as a loading control. (I to L) Cell invasion in cells transfected with GIT2 shrna (I and K) or CYTH3 shrna (J and L), as assessed by Transwell invasion assays. (M to R) Proliferation and colony forming ability in cells transfected with GIT2 shrna (M, O and Q) or CYTH3 shrna (N, P and R), as assessed by BrdU incorporation and soft agar assays, respectively. Data are means ± SD from 3 experiments; blots are representative. N.S, not significant; *P<0.05, **P<0.01, analyzed by Student s t-test.

6 Fig. S5. Luciferase assay results. (A) Activity of Git2 pmirtarget luciferase reporter plasmids in TA2-C7 cells co-transfected with mir-200b/c mimics, mir-200b/c mimics and full-length H19 plasmids (ph19), or mir-200b/c mimics and a mir- 200b/c binding site-mutant H19 plasmids (ph19mut1) were. (B and C) Abundance of mir-200b/c (B) or let-7b (C) in C13-PT, C13-PB and C13-LM cells transfected with H19 sirna (#2 sirna) or a control. (D to G) Relative amount of GTP-bound ARF6 (D and F; assessed by GST-GGA3 pulls down analysis) or E-cadherin (E and G) in C13-PT, C13-PB and C13-LM cells transfected with Git2 cdna mutated at mir- 200b/c binding sites (D and E), Cyth3cDNA mutated at let-7b binding sites (F and G), or the respective control plasmids. Data are means ± SD (n=3); N.S, not significant; **P<0.01, ***P<0.001.

7 Table S1. Metastases resulting from orthotopic injection of cells from distinct clones. The numbers of circulating tumor cells, metastatic lesions of TA1 mice 4 weeks after orthotopic injection of cells isolated from single mouse mammary tumor that arose spontaneously without chemical stimulus in a wild-type TA2 mouse. Clone ID Cells in blood Pulmonary tumor lesions Extra-pulmonary tumor lesions Clone Clone Clone Clone ± ± ±2.40 Clone ± Clone Clone ± Clone Clone ± Clone ± ± ±2.45 Clone Clone ± ± ±1.97 Clone Clone Clone Clone ±

8 Table S2. Clinical characteristics of MBC patients. The clinical stages and subtypes of 60 patients with metastatic breast cancer were evaluated by TNM system and pathologic feature. IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ER, estrogen receptor; PR, progesterone receptor; HER, human epidermal growth factor receptor. T T1 21(35.0%) T2-T4 39(65.0%) N N1 19(31.7%) N2-N3 41(68.3%) Histology IDC 47(78.3%) ILC 11(18.3%) other 2(3.3%) Grading G1 2(3.3%) G2 26(43.3%) G3 32(53.3%) ER ER - 18(30%) ER + 42(70%) PR PR - 21(35.0%) PR + 39(65.0%) HER HER2-48(80%) HER2 + 12(20%)

9 Data file S1. Microarray results. The raw microarray data is available on Dryad. Here, the normalized microarray data were further sorted by pair comparison between non-metastatic clones (Group N), weakly metastatic clones (Group W) and highly metastatic clones (Group H). Data are provided in an Excel (.xls) file in the auxiliary files online. Data file S2. mirna and H19 sequences. Online searching for the mouse mir- 200b/c, Let-7b and H19 sequences that were presented 5-3. >mmu-mir-200b-3p MIMAT : UAAUACUGCCUGGUAAUGAUGA >mmu-mir-200c-3p MIMAT : UAAUACUGCCGGGUAAUGAUGGA >mmu-let-7b-5p MIMAT : UGAGGUAGUAGGUUGUGUGGUU >mmu-h19, Accession ID AK145379: GGTTGGGGGGTATCGGGGAAACTGGGGAAGATGGGAGAGCTGGAGGAGAGTCGTGGGGT CCGAGGAGCACCTCGGCATCTGGAGTCTGGCAGGAATGTTGAAGGACTGAGGGGCTAGCT CAGGCAGAGCAAAGGCATCGCAAAGGCTGGAAAACATCGGAGTGAAGCTGAAGGGCCTG AGCTAGGGTTGGAGAGGAATGGGGAGCCAGACATTCATCCCGGTTACTTTTGGTTACAGG ACGTGGCGGCTGGTCGGATAAAGGGGAGCTGCTGGGAAGGGTTCGACCCCAGACCTGGG CAGTGAAGGTATAGCTGGCAGCAGTGGGCAGGTGAGGACCGCCGTCTGCTGGGCAGGTG AGTCTCCTTCTTCTCTCTTGGCCTCGCTCCACTGACCTTCTAAACGAAGGTTTAGAGAGGG GGCCTGGTGAGAAGAAGCGGCTGGCCTCGCAGCAGAATGGCACATAGAAAGGCAGGATA GTTAGCAAAGGAGACATCGTCTCGGGGGGAGCCGAGACAGAAGGAGGCTGGGGGACCAT TGGCGACCCCAGGTGGAAAGAGCTCTTAGAGAGAAGAAAGAAGAGGTGCAGGGTTGCCA GTAAAGACTGAGGCCGCTGCCTCCAGGGAGGTGATAGGAGTCCTTGGAGACAGTGGCAG AGACCATGGGATCCAGCAAGAACAGAAGCATTCTAGGCTGGGGTCAAACAGGGCAAGAT GGGGTCACAAGACACAGATGGGTCCCCAGCCGCCACAACATCCCACCCACCGTAATTCAC TTAGAAGAAGGTTCAAGAGTGGCTCTGGCAAAGTCCCAAGTTTGCCAGAGCCTCAATAAC TGGAGAATGGAAAAGAAGGGCAGTGCAGGGTGTCACCAGAAGGGGAGTGGGGGCTGCAG GTATCGGACTCCAGAGGGATTTTACAGCAAGGAGGCTGCAGTGGGTCCAGCCTGCAGACA CACCATTCCCATGAGGCACTGCGGCCCAGGGACTGGTGCGGAAAGGGCCCACAGTGGACT TGGTACACTGTATGCCCTAACCGCTCAGTCCCTGGGTCTGGCATGACAGACAGAACATTTC CAGGGGAGTCAAGGGCACAGGATGAAGCCAGACGAGGCGAGGCAGGCGGGGCAGAATG AATGAGTTTCTAGGGAGGGAGGTTGGGTGCAGGTAGAGCGAGTAGCTGGGGTGGTGAGC CAGGGAGGCACTGGCCTCCAGAGTCCGTGGCCAAGGAGGGCCTTGCGGGCGGCGACGGA GCAGTGATCGGTGTCTCGAAGAGCTCGGACTGGAGACTAGGCCAGGTCTCCAGCAGAGGT GGATGTGCCTGCCAGTCACTGAAGGCGAGGATGACAGGTGTGGTCAATGTGACAGAAAG ACATGACATGGTCCGGTGTGATGGAGAGGACAGAAGGGCAGTCATCCAGCCTTCTTGAAC ACCATGGGCTGGCGCCTTGTCGTAGAAGCCGTCTGTTCTTTCACTTTTCCCAAAGAGCTAA CACTTCTCTGCTGCTCTCTGGATCCTCCTCCCCCTACCTTGAACCCTCAAGATGAAAGAAA TGGTGCTACCCAGCTCATGTCTGGGCCTTTGAATCCGGGGACTTCTTTAAGTCCGTCTCGTT CTGAATCAAGAAGATGCTGCAATCAGAACCACTACACTACCTGCCTCAGGAATCTGCTCC AAGGTGAAGCTGAAAGAACAGATGGTGTCAACATTTTGAAAGAGCAGACTCATAGCACCC ACCCACCCCTGAGAATCCATCTTCATGGCCAACTCTGCCTGACCCGGGAGACCACCACCC ACATCATCCTGGAGCCAAGCCTCTACCCCGGGATGACTTCATCATCTCCCTCCTGTCTTTTT CTTCTTCCTCCTTTCCTGTAATTCTGTTTCTTTCCTTTTGTTCCTTCCTTGCTTGAGAGACTC AAAGCACCCGTGACTCTGTTTCCCCATTTACCCCCTTTTGAATTTGCACTAAGTCGATTGCA CTGGTTTGGAGTCCCGGAGATAGCTTTGAGTCTCTCCGTATGAATGTATACAGCGAGTGTG TAAACCTCTTTGGCAATGCTGCCCCAGTACCCACCTGTCGTCCATCTCCGTCTGAGGGCAA CTGGGTGTGGCCGTGTGCTTGAGGCCTCGCCTTCCCCTCGCCTAGTCTGGAAGCAGTTCCA TCATAAAGTGTTCAACATGCCCTACTTCATCCTTTGCCCCTCCTCACCAGGGCCTCACCAG AGGTCCTGGGTCCATCAATAAATACAGTTACAGCC

10 Data file S3. mirna target information. The predict target sites and exact positions of mouse H19 for mir-200b/c, let-7b were analyzed by online RNAhybrid tool. target : mmu-h19 length: 2257 mirna : mmu-mir-200b-3p length: 22 mfe: kcal/mol position 339 target 5' C G 3' CGUC UGCUGGGCAG GUAG AUGGUCCGUC mirna 3' A UA AUAAU 5' target: mmu-h19 length: 2257 mirna : mmu-mir-200c-3p length: 23 mfe: kcal/mol position 338 target 5' G G G 3' CCGUC UGCU GGCAG GGUAG AUGG CCGUC mirna 3' A UA G AUAAU 5' target: mmu-h19 length: 2257 mirna : mmu-let-7b-5p length: 22 mfe: kcal/mol position 1637 target 5' G U A C G 3' AACCAC AC CUA CUGCCUCA UUGGUG UG GAU GAUGGAGU mirna 3' UGU 5'

11 Data file S4. ArfGEF target information. The screen shot of let-7b target sites on 10 ArfGEF-encoding genes. 1. BIG1 (ARFGEF1) 2. BIG2 (ARFGEF2) 3. GBF1 4. BRAG1 ( IQSEC2) 5. BRAG2 (IQSEC1) 6. CYTH1 7. EFA6A (PSD)

12 8. PSD2 9. FBXO8 10. CYTH3

13 Data file S5. sirna, shrna, and PCR primer sequences. The sequences for commercially provided or synthesized oligonucleotides. SiRNAs: 27mer H19 sirna duplexes: ShRNAs: H19 shrna: #1. V3SM GACGGCTTCTACGACAAGG #2. V3SM TTCAGAACGAGACGGACTT #3. V3SM TTATCCGACCAGCCGCCAC Git2 shrna: #1. V3SM GTCAACTTCGTCGTACACA #2. V3SM TGGACGAACGCTAACATCT #3. V3SM TCATGACCTAAGCACTTGG CYTH3 shrna: #1. V3SM CAGCAGGTCGTTCTCAATT #2. V3SM GAACGTGTGTGTCAGGTCA #3. V3SM CTGACATTTACCTATTTCT Primers: ph19mut1: Forward: AAGCTTCAGCAGAATGGCACATA Reverse: CTCGAGGGAGGCTGGGGGACCAT ph19mut2: Forward: AAGCTTCGTTCTGAATCAAGA Reverse: CTCGAGCCACTACACTACCTGCCT Git2 mir-200b/c mutant: Forward: GCGGATCCCTGGACCCTGCCGC Reverse: GACCATGGAGG CGGAAAGCTATT CYTH3 let-7b mutant: Forward: GCGGATCCTATGAAAGTCAAGCG Reverse: GACCATGGTAAGATCCCAAGCTTAC Oligonucleotides: psicheck2-mir-200b 4X: GCAAGCTTAGGCCGTCGTGCTGGGCAGGCCGTCGTGCTGGGCAGGCCGTCGTGCTGGGCA GGCCGTCGTGCTGGGCAGGCGGCCGCAAGAGCTC psicheck2-mir-200b 4X: GCAAGCTTGGCCGTCGTGCTGGGCGGCCGTCGTGCTGGGCGGCCGTCGTGCTGGGCGGCC GTCGTGCTGGGCGAGCTC psicheck2-let-7b 4X:

14 GAACCACTACACTACCTGCCTCAGGCGGCTCGAGGAACCACTACACTACCTGCCTCAGGA ACCACTACACTACCTGCCTCAGGAACCACTACACTACCTGCCTCAGGAACCACTACACTAC CTGCCTCAG GCGGCCGCAA