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(Ng/Ml) - 10 100 E2 - 10 100 E2 1 1.8 2 6.2 7.5 7.2 WT1 K303R1 ER B WT1 K303R2 - Lep - Lep NC A Cyclin D1 1 2.4 2 3.5 pS2 ** ** 1 2.5 2.2 4.7 ** Cathepsin D 1 1.9 2 4.6 36B4 ** ** n.s. C WT P K303R P EREluc Activity (RLU) Lep (min) - 5 10 - 5 10 pS118 1 1.4 1.5 3 3.5 3.3 pS167 Lep (ng/ml) - 10 100 E2 - 10 100 E2 1 1.8 2 6.2 7.5 7.2 WT1 K303R1 ER Supplementary Figure 1. Activation of K303R ER receptor by leptin. A, ER-transactivation assay in cells treated with vehicle (-), Lep at 10 and 100 ng/ml or 17- estradiol (E2 10nM, positive control). Data are reported as Relative Lights Units (RLU). n.s.=nonsignificant, **P<0.01. B, mRNA expression of Cyclin D1, pS2, Cathepsin D and 36B4 (internal standard) by RT-PCR in cells treated with vehicle (-) or Lep 100ng/ml for 24 hours. NC, negative control. Numbers represent the average fold change between Cyclin D1, pS2 or Cathepsin D and 36B4 levels. C, Total extracts from cells treated with Lep 100ng/ml for 5 and 10 min were analyzed for phosphorylation of serines 118 and 167 (pS118 and pS167) and expression of ER by immunoblot analysis. Numbers below the blots represent the average fold change between pER levels and ER total protein expression. A B * * * ** n.s. ** * * ) n.s. * 4 n.s. * * n.s. MTT Assay (Fold Change) Cell Count (Cell # x10 Lep (ng/ml) - 10 100 - 10 100 WT1 K303R1 Lep (ng/ml) - 10 100 - 10 100 - 10 100 - 10 100 - 10 100 1 2 P 1 2 WT K303R Supplementary Figure 2. Leptin effects on anchorage-dependent growth. A, MTT growth assay in cells treated with vehicle (-), or Lep at 10 and 100ng/ml for 4 days. n.s.=nonsignificant, *P<0.05. B, Trypan blue-exclusion proliferation assay in cells treated as indicated for 4 days. ns=nonsignificant, *P=0.01, **P<0.005. AB MCF-7 SKBR3 CAFs ObRl MCF-7 CAFs NC ObRs Ob ER 36B4 ER GAPDH Supplementary Figure 3. Characterization of cancer-associated fibroblasts (CAFs). A, RT-PCR for leptin (Ob) and 36B4 (internal standard) mRNA expression. NC, negative control. B, Immunoblotting analysis of whole-cell lysates from MCF-7, SKBR3 and CAFs cells for ObR long and short isoforms, ER and ER expression. GAPDH was used as a control for equal loading and transfer. A D WT K303R SKBR3 P YFP-ER GAPDH K303R WT B *** Lep (ng/ml) - 10 100 1000 E ** * * * *** (Colony #) Soft Agar Assay C ** FM CM CM+LepAb FM CM CM+LepAb * WT K303R * F ** (Colony #) Soft Agar Assay ** ** n.s. K303R WT Lep (ng/ml) - 10 100 1000 - 10 100 1000 FM CM CM+LepAb WT K303R Supplementary Figure 4. Leptin increases growth and migration in K303R ER SKBR3 cells. A, Immunoblotting analysis for ER and GAPDH in YFP-WT and YFP-K303R ER stable transfected SKBR3 pools. B, Real-time RT-PCR for ObRl and ObRs. ***P=0.0001. C, Soft agar and D, in vitro scratch assays in cells treated as indicated. E, Soft agar and F, in vitro scratch assays in cells treated with full media (FM), CAFs-derived conditioned media (CM) or leptin-depleted CM (CM+LepAb). n.s.=nonsignificant, *P<0.05, **P<0.005. Small squares represent time 0 of treatment. Supplementary Table I. Oligonucleotide primers used in this study. Gene Name Gene Primer Sequences Symbol Fibroblast activation protein FAP Forward 5’-AGAAAGCAGAACTGGATGG-3’ Reverse 5’-ACACACTTCTTGCTTGGAGGAT-3’ 36B4 36B4 Forward 5’-CTCAACATCTCCCCCTTCTC-3’ Reverse 5’- CAAATCCCATATCCTCGT -3’ Leptin Receptor Long ObRl Forward 5’-GATAGAGGCCCAGGCATTTTTTA-3’ Isoform Reverse 5’- ACACCACTCTCTCTCTTTTTGATTGA-3’ Leptin Receptor Short ObRs Forward 5’- ATTGTGCCAGTAATTATTTCCTCTTCC-3’ Isoform Reverse 5’- CCACCATATGTTAACTCTCAGAAGTTCAA-3’ Chemokine (C-X-C motif) CXCR4 Forward 5’-AATCTTCCTGCCCACCATCT-3’ receptor 4 Reverse 5’-GACGCCAACATAGACCACCT-3’ Insulin Receptor IR Forward 5’-AGGAGCCCAATGGTCTGA-3’ Reverse 5’-AGACGCAGAGATGCAGC-3’ Interleukin 2 Receptor IL2R Forward 5’-GGCCATGGCTGAAGAAGGT-3’ Reverse 5’-CTGGACGTCTCCTCCATGCT-3’ Interleukin 6 Receptor IL6R Forward 5’-TGAGCTCAGATATCGGGCTGAAC-3’ Reverse 5’-CGTCGTGGATGACACAGTGATG-3’ Epidermal growth factor EGFR Forward 5’-GGACTCTGGATCCCAGAAGGTG-3’ receptor Reverse 5’-GCTGGCCATCACGTAGGCTT-3’ Insulin growth factor-1 IGF1R Forward 5’-CACGACGGCGAGTGCAT-3’ receptor Reverse 5’-ACAGACCTTCGGGCAAGGA-3’ Fibroblast growth factor FGFR3 Forward 5’-TGCTGAATGCCTCCCACG-3’ receptor 3 Reverse 5’-CGTCTTCGTCATCTCCCGAG-3’ Estrogen receptor ERα Forward 5’-TGATTGGTCTCGTCTGGCG-3’ Reverse 5’-CATGCCCTCTACACATTTTCCC-3’ Cyclin D1 CD1 Forward 5’-TCTAAGATGAAGGAGACCTATC-3’ Reverse 5’-GCGGTAGTAGGACAGGAAGTT-3’ 18s rRNA 18s Forward 5’-CCCACTCCTCCACCTTTGAC-3’ Reverse 5’-TGTTGCTGTAGCCAAATTCGTT-3’ Cathepsin D CD Forward 5’-AACAACAGGGTGGGCTTC-3’ Reverse 5’-TTTGAGTAGTCAAAGTCAGAGC-3’ Trefoil factor 1/pS2 pS2 Forward 5’-TTCTATCCTAATACCATCGACG-3’ Reverse 5’-TTTGAGTAGTCAAAGTCAGAGC-3’ Leptin Ob Forward 5’-GAGACCTCCTCCATGTGCTG-3’ Reverse 5’-TGAGCTCAGATATCGGGCTGAAC-3’ Epidermal growth factor EGF Forward 5’-CCCTAAGTCGAGACCGGAAGT-3’ Reverse 5’-CAAGAGTACAGCCATGATTCCAAA-3’ Interleukin 6 IL6 Forward 5’-CCAGGAGCCAGCTATGAAC-3’ Reverse 5’-CCCAGGGAGAAGGCAACTG-3’ Insulin INS Forward 5’-TCAGAAGAGGCCATCAAGCA-3’ Reverse 5’-AGATGCTTCACGAGCCCAGC-3’ Supplementary Table II Unchanged genes in the receptor family of CAFs-secreted factors among WT and K303R ERα-expressing MCF-7 breast cancer cells. Gene name Gene Symbol Interleukin 2 receptor , interleukin 2 receptor γ, interleukin 7 receptor, IL2RA, IL2RG, IL7R, interleukin 8 receptor β, interleukin 10 receptor , interleukin 12 receptor IL8RB, IL10RA, β2, interleukin 13 receptor 2, interleukin 18 receptor 1, interleukin 22 IL12RB2, IL13RA2, receptor IL18R1, IL22RA Colony stimulating factor 3 receptor, colony stimulating factor 2 receptor CSF3R, CSF2R Platelet derived growth factor receptor β PDGFRB Nerve growth factor receptor NGFR Tumor necrosis factor receptor, member 8, 9, 10 and 17 TNFR8, TNFR9, TNFR10, TNFR17 Chemokine (C-X-C motif) receptor 3, chemokine (C-X-C motif) receptor CXCR3, CXCR6, 6, chemokine (C-C motif) receptor 2, chemokine (C-C motif) receptor 3, CCR2, CCR3, CCR5, chemokine (C-C motif) receptor 5, chemokine (C-C motif) receptor 6 CCR6 Representative probesets from pathway analysis showing unchanged gene expression in the receptors for CAFs-derived soluble factors among K303R-expressing cells and WT cells. The specific requirement were: genes for which no probeset for that gene was different between the control and K303R by p<0.2, and genes for which no probeset for that gene was different between the control and K303R by 0.25 fold change. Supplementary Table III Selection of relevant up-regulated genes in the leptin signaling pathway in K303R ER-expressing MCF-7 breast cancer cells. Gene name Gene Symbol Parametric P-value Fold change in K303R clones Janus Kinase 2 JAK2 <1e-07 3,7 Signal Transducer and STAT <1e-07 15,1 Activator of Transcription v-fos FBJ Murine FOS 4e-07 5,2 Osteosarcoma Viral Oncogene Homolog Suppressor of Cytokine SOCS3 0.009 1,2 Signaling 3 Representative probesets from pathway analysis showing gene expression changes in the leptin signaling along with the p-value and the fold change of K303R-expressing cells compared to WT cells studied by microarray analysis. In cases where the same genes were deemed significant across multiple probesets, only one is shown. .
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