Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 1. LEFTY1 Expression Is Restricted to a Subset of Luminal Cells and Rare Basal Cells, while BMP7 Is Secreted Predominantly by Basal Cells (A) Left: representative FACS plot of the mouse mammary epithelial cell subpopulations stained with the indicated cell-surface markers. Different subpopulations are indicated as described in the color key. Right: Principal-component analysis reveals different subpopulations based on single-cell PCR analysis. (B) Hierarchically clustered analysis of single-cell PCR analysis of sorted mouse mammary epithelial populations. (C) Representative images of BMP7 protein staining of mammary glands isolated from 6- to 8-week-old WT mice. Scale bar, 20 mm. (D) Representative images of LEFTY1 protein staining of mammary glands isolated from 6- to 8-week-old WT mice. Scale bar, 20 mm. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Expressing, Staining, Isolation

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 2. LEFTY1 Induces and BMP7 Reduces Ductal Branching and the Long-Term Proliferative Potential of Mammary Progenitor Cells (A) Schematic representation of procedure used to inject ligand-secreting L1 fibroblasts into the mammary fat pad followed by analysis of mammary glands. (B) Quantification of branches counted in glands exposed to the LEFTY1 (n = 6) or control (n = 7) group. Statistical analysis, Student’s t test. ***p < 0.001. Data are represented as mean ± SEM. (C) Quantification of branches counted in glands exposed to the BMP7 (n = 6) or control (n = 7) group. Statistical analysis, Student’s t test. *p < 0.05. Data are represented as mean ± SEM. (D) Representative images of the whole-mount staining of the indicated groups of mice. Magnification, 403. (E) Representative images of the organoids grown in the presence of BMP7, LEFTY1, or control (n = 7). Scale bar, 100 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Control, Staining

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 3. LEFTY1 Simultaneously Suppresses SMAD2 and SMAD5 in Basal Cells (A) Quantification of pSMAD2-positive cells within the luminal and basal compartments of endogenous ductal structures exposed to LEFTY1-secreting fibro- blasts. Data are represented as mean ± SD. Statistical analysis, Student’s t test. ****p < 0.0001. (B) Quantification of pSMAD2-positive cells within the luminal and basal compartments of endogenous ductal structures exposed to BMP7-secreting fibroblasts. Data are represented as mean ± SD. Statistical analysis, Student’s t test. n.s., non-significant. (C) Quantification of pSMAD5-positive cells within the luminal and basal compartments of endogenous ductal structures exposed to LEFTY1-secreting fibro- blasts. Data are represented as mean ± SD. Statistical analysis, Student’s t test. *p < 0.05; ****p < 0.0001. (D) Quantification of pSMAD5-positive cells within the luminal and basal compartments of endogenous ductal structures exposed to BMP7-secreting fibroblasts. Data are represented as mean ± SD. Statistical analysis, Student’s t test. *p < 0.05; n.s., non-significant. (E) Representative images of the quantified sections for pSMAD2 analysis of mammary glands exposed to LEFTY1-secreting fibroblasts. (F) Representative images of the quantified sections for pSMAD5 analysis of mammary glands exposed to LEFTY1-secreting fibroblasts. (G) Representative images of the quantified sections for pSMAD2 analysis of mammary glands exposed to BMP7-secreting fibroblasts. (H) Representative images of the quantified sections for pSMAD5 analysis of mammary glands exposed to BMP7-secreting fibroblasts. (I) Western blot for pSMAD2, pSMAD5, total SMAD2/3, or SMAD1/5/8 proteins in COMMA-D cells blocked with LEFTY1 and stimulated with BMP7 and Nodal (n = 3). (J) Quantification of the pSMAD5 signal detected by western blot in the indicated conditions. (K) Quantification of the pSMAD2 signal detected by western blot in the indicated conditions. Scale bar, 20 mm. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Western Blot

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 4. LEFTY1 Directly Binds to the BMP Receptor BMPR2 (A) Relative mRNA levels of Bmpr2 in basal cell populations (CD49fhiCD24med and CD49flowCD24low) and luminal ones (CD49flowCD24hi and CD49flow/-CD24med). Statistical analysis is one-way ANOVA with Dunnett’s adjustment. (B) Representative images of proximity ligation assay show the interaction between LEFTY1 and BMPR2. Red puncta indicate positive interactions. Scale bar, 10 mm. (C) Quantification of the positive interaction between BMP7 or LEFTY1 with BMPR2 (n = 3). Statistical analysis is one-way ANOVA with Dunnett’s adjustment. ****p < 0.0001. (D) Western blot on co-immunoprecipitated beads of LEFTY1 and BMPR2. (E) Schematic representation of the mechanism by which LEFTY1 inhibits SMAD5 and SMAD2 phosphorylation. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Phospho-proteomics

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 5. Attenuated LEFTY1 Abrogates the Proliferation of Breast Cancer Cells In Vivo (A) Percentage of genetic alterations in LEFTY1 found in METABRIC (indicated in blue) and TCGA (indicated in red) human breast cancer datasets. (B) Characterization of LEFTY1 copy number in five breast cancer PDX models. (C) Characterization of LEFTY1 mRNA levels in five breast cancer PDX models. (D) Characterization of LEFTY1 protein levels by immunofluorescence in five breast cancer PDX models. Scale bar, 100 mm. (E) Western blot analysis shows the knockdown efficiency of the shLEFTY1 #2 (shL2) and shLEFTY1 #3 (shL3) virus measured by decrease in LEFTY1 protein. (F) Size of the tumors arising from breast tumor cells infected with two viruses that knock down LEFTY1 (shL2 and shL3). Each circle represents a single tumor normalized to red fluorescent protein percentage (RFP%) as elaborated in Figure S6A. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: In Vivo, Western Blot, Knockdown, Virus, Infection

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 6. Autocrine LEFTY1 Is Necessary for the Proliferation Tumor-Initiating Breast Cancer Cells (A) LEFTY1 mRNA expression levels in the tumorigenic (TG) and non-TG (NTG) populations sorted based on CD49f and EpCAM expression. The populations were sorted from five different PDX models (n = 2–3 tumors per PDX model).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Expressing

Journal: Cell stem cell

Article Title: LEFTY1 Is a Dual-SMAD Inhibitor that Promotes Mammary Progenitor Growth and Tumorigenesis.

doi: 10.1016/j.stem.2020.06.017

Figure Lengend Snippet: Figure 7. LEFTY1 Binds to BMPR2 and Is a Concomitant SMAD2 and SMAD5 Inhibitor in Tumor-Initiating Cells (A) Representative pictures of cells from isolated tumorigenic (TG) and non-TG (NTG) cells to assess LEFTY1-BMPR2 and BMP7-BMRP2 interaction. Scale bar, 20 mm. (B) Quantification by proximity ligation assay of LEFTY1-BMPR2 or BMP7-BMPR2 interaction in TG cells or NTG sorted cells. (C) Representative images of cells from isolated TG and NTG cells stained to assess pSMAD5 staining. Scale bar, 100 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant EGF Thermo Fisher Scientific Cat# PMG8043 Recombinant Noggin R&D Systems Cat# 6057-NG Growth Factor Reduced Matrigel BD Biociences Cat# 356230 Matrigel BD Biociences Cat# 356234 LEFTY-1 R&D Systems Cat# 994-LF BMP7 R&D Systems Cat# 5666-BP BMP2 R&D Systems Cat# 355-BM BMP4 R&D Systems Cat# 314-BP CRIPTO-1 R&D Systems Cat# 1538-CR NODAL R&D Systems Cat# 1315-ND Recombinant Human TGF-beta 1 Protein R&D Systems Cat#240-B-002 DAPI Sigma-Aldrich Cat# 32670 Dispase StemCell Technologies Cat#07913 Collagenase/Hyaluronidase StemCell Technologies Cat #07912 DNase I solution StemCell Technologies Cat#07900 Lefty Blocking Peptide Santa Cruz Biotechnolgy Cat# sc-365845 P LDN-193189 Selleckchem Cat# S2618 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat#E1910 DUOLink Sigma Cat#921021 Pierce CO-IP Kit Thermo Fisher Scientific Cat#26147 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216, RRID:CVCL_0063 MDA-MD-157 ATCC Cat# HTB-24, RRID:CVCL_0618 3T3-L1 feeder cells Kindly provided by Dr. Nusse N/A L1-wnt3a feeder cells Kindly provided by Dr. Nusse N/A MDA-MB-231 ATCC Cat# HTB-26, RRID:CVCL_0062 COMMA-D beta cells Kindly provided by Dr. Medina Campbell et al., 1988 Experimental Models: Organisms/Strains C57BL/6 mice Jackson Laboratory Cat# JAX:000664, RRID:IMSR_JAX:000664 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Jackson Laboratory Cat# JAX:005557, RRID:IMSR_JAX:005557 pCx-GFP Kind gift of Dr. Irving Weissman N/A Oligonucleotides Cloning primers This paper Table S6 qRT-PCR primers This paper Table S6 Copy number determination primers This paper Table S6 Single cell assay ID This paper Table S3 Recombinant DNA pEIZ-HIV-ZsGreen Kind gift of Dr. Zena Werb N/A HIV-Lefty1-ZsG This paper N/A HIV-Che Shimono et al., 2009 N/A HIV-Bmp7-Che This paper N/A pGL3 BRE-luciferase Kind gift of Martine Roussel and Peter ten Dijike, Addgene #45126 pRL-TK Renilla luciferase vector Promega Cat# E2261 pEGFP-C3 (discontinued) Clontech Clontech Bmp7 cDNA clone Origene Cat# MC201085 clone pSICO-R Addgene Cat#12084 Lefty1 cDNA clone IMAGE Cat# 5221120 plasmid clone (Continued on next page) e2 Cell Stem Cell 27, 1–16.e1–e8, August 6, 2020

Techniques: Isolation, Proximity Ligation Assay, Staining