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Supplementary Materials Supplementary Figures and Tables Figure S1. Validation of EMT-selective small molecules (A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square), and HMLE_Twist (black diamond) cells treated with various concentrations of Cmp302, Cmp308, and Dev4 for 3 days. Cell survival was determined using an ATP-based luminescence assay. (B) Dose response curves of HMLER_shGFP (blue circle), HMLER_shEcad (red square), and HMLER_Twist (black diamond) cells treated with various concentrations of Cmp302, Cmp308, and Dev4 for 3 days. Cell survival was determined using an ATP- based luminescence assay. (C) Representative fluorescence images of HMLE_shGFP_dsRed and HMLE_Twist_GFP cells mixed in 1:1 ratio and treated with 5 nM paclitaxel, 2 μM Cmp302, 2 μM Cmp308 or DMSO control for 5 days. Scale bar: 50 μm (D) Proportion of HMLE_shGFP_dsRed and HMLE_shEcad_GFP cells that were mixed in a 1:1 ratio and treated with doxorubicin or Dev4 at indicated concentrations for 5 days. (E) Molecular structure of Dev2. (F)Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square), and HMLE_Twist (black diamond) cells treated with various concentrations of Dev2 for 3 days. Cell survival was determined using an ATP-based luminescence assay. (G) Representative fluorescence images of MDA-MB-157 cells either cultured under suspension conditions for 36 h and transferred onto adherent cell culture plates for 24h, 1 or maintained in standard adherent cell culture. Cells were stained using FITC-conjugated phalloidin to visualize F-actin fibers. Scale bar: 50 μm. (H) Western blot analysis of epithelial (CK8/18, E-cadherin) and mesenchymal (fibronectin) markers of cell lysates from HMLER_shCtrl, HMLER_Twist and MDA- MB-157 cells grown under either adherent or suspension conditions. (I) Flow cytometric analysis of MDA-MB-157 cells, cultured under either adherent or suspension conditions for 36 h, using CD24 and CD44 antibodies. (J) MDA-MB-157 cells in suspension were treated with increasing concentrations of Cmp308 or paclitaxel for 36 h and analyzed for CD44 and CD24 expression by flow cytometry. The numbers of CD44hiCD24lo and CD44loCD24hi cells were quantified and normalized relative to DMSO controls. (K) Dose-response curves of MDA-MB-157 cells cultured under adherent (blue circle) or suspension (red diamond) conditions and treated with specified concentrations of paclitaxel, Cmp302, or Cmp308 for 3 days. Cell survival was determined using an ATP- based luminescence assay. Figure S2. EMT increases sensitivity to ER stressors (A) Western blot analysis of HMLER_shGFP and HMLER_Twist cells treated with vehicle (DMSO) or increasing concentrations of Dev4 or thapsigargin for 12 h and probed for ATF4. (B) Dose-response curves of HMLE_shGFP, HMLE_shEcad, and HMLE_Twist cells treated with increasing concentrations of tunicamycin, thapsigargin, DTT, and A23187 for 3 days. Cell survival was determined using an ATP-based luminescence assay. 2 (C) Dose-response curves of Panc1cells, pretreated with or without10 ng/ml TGFb for 3 or 8 days, treated increasing concentrations of tunicamycin and thapsigargin for 3 days. Cell survival was determined using an ATP-based luminescence assay. (D) Western blot analysis for intact and cleaved Caspase-3 expression in non-EMT (HMLER_shGFP) and EMT (HMLER_Twist) cells treated with increasing concentrations of thapsigargin. (E) IC50 for four luminal breast cancer cells, MCF7, MDA361, T47D and ZR-75-3(blue dots), and six basal-B lines, BT549, 4T1, Hs578T, MDA231, MDA436 and MDA157 (red dots) treated with various concentrations of tunicamycin, thapsigargin, DTT or A23187 for 3 days. (F) MCF7, ZR75-3, MDA231 and 4T1 cells were treated with solvent control, 0.1 µg/ml Tunicamycin (TM), or 10 nM Thapsigargin (TG) for 3 days. Cells were fixed with 70% ethanol, treated with RNase-A, and stained with propidium iodide. Fraction of cells in sub-G1 phrase was analyzed by FACS. Figure S3. Cells that undergo an EMT are highly secretory (A) Gene Set Enrichment Analysis for genes differentially expressed between control HMLE cells and HMLE cells induced into an EMT state with Gsc, shEcad, Snail, TGFb, or Twist. The Normalized Enrichment Score for each comparison is plotted and indicates strong overexpression in EMT cells of genes encoding ECM components (upper panel) and Collagens (lower panel). 3 (B) Log2 fold-change in the expression of 20 secretory pathway component genes in HMLE cells induced into an EMT state with Gsc, shEcad, Snail, TGFb or Twist relative to controls. The genes tested were 20 SPCGs (secretory pathway capacity gene), including SARA1, Sec24D, Sec23A, KDELR3, Sec13L1, Sec61A1, Sec61A2, COPZ2, Sec14L1, Sec14L2, COPB2, TRAM2, COPG, ARCN1, Sec31L1, TRAM1, SRPRB, SRP54, Sec24A and KDELR1. (C) Western blotting showing the expression of Sec16-GFP and GAPDH in HMLE_Ctrl and HMLE_shEcad cells transfected with Sec16-GFP construct. (D) Autoradiograph showing 35S-methionine/cysteine-labeled secreted proteins from HMLE control and HMLE_shEcad cells, pretreated with or without 5 μg/ml of Brefeldin A. Secreted proteins were harvested at the indicated time points. Figure S4. Inhibition of ECM genes abrogates migration and ER stress sensitivity (A) 2×105 HMLE_shEcad cells infected with shLuc or DK-1 hairpins (FN-1 and PAI-1) were seeded onto a 6-well plate with serum free, BPE free culture medium. Conditional media collected at 6 hour or 16 hour post cell seeding were analyzed by PAGE, and the secreted protein from each sample were detected by silver stain. (B) Migratory ability of HMLE_shEcad_shLuc and HMLE_shEcad_DK-1 cells was measured using an in vitro transwell assay (10 hour). Representative images were shown. (C) qPCR analysis for expression of GADD34 and CHOP in HMLE_shEcad_shLuc and HMLE_shEcad_DK-1 cells treated with increasing concentrations of Dev4, thapsigargin, or DMSO solvent for 6 h. 4 Figure S5. BiP inhibition activates the UPR in basal-B but not luminal breast cancer cells Expression of UPR pathway components in three luminal cells, MCF7, T47D and ZR75- 3, and four basal-B cells, MDA157, Hs578T, MDA231 and BT549 infected with control (shLuc) or BiP specific hairpins (shBiP) were shown. Expression of phospho-eIF2α, total eIF2α and tubulin was detected by western blotting. XBP1 and XBP1 splice variant was analyzed by RT-PCR. Figure S6. EMT activates PERK-eIF2a but not IRE1-XBP1 (A) RT-PCR analysis of XBP1 expression in HMLE and HMLER cells induced into EMT (HMLE_Twist, HMLE_shEcad, HMER_Twist, HMLER_shEcad), their non-EMT controls (HMLE_shGFP, HMLER_shGFP), and non-EMT luminal human breast cancer cell lines, as well as in EMT basal-B breast cancer cell lines. (B) Western blotting showing the expression of markers for EMT and UPR in Panc1 cells treated with 10 ng/ml TGFb for 0, 2, 4, 5, 6, 7 and 8 days. (C) HMLE_shGFP and HMLE_shEcad cells were pretreated with 0, 0.5 µM, 1 µM or 2 µM of PERK inhibitor for 24 hours, then treated with or without 40 nM thapsigargin for another 2 hours prior to protein extraction. Activity of PERK-eIF2alpha pathway was measured by Western blotting. Figure S7. Effects of PERK inhibitor on cellular proliferation (A) 1×105 HMLE_shGFP and HMLE_shEcad cells, which have been pretreated with DMSO or 1 μM PERK inhibitor for 2 days, were placed with or without 1 μM PERK 5 inhibitor for 4 days. Relative cell growth was measured by counting with a hemacytometer. (B) 1×105 HMLE_shGFP and HMLE_shEcad cells, which have been pretreated with DMSO or 1 μM PERK inhibitor for 2 days, were placed without PERK inhibitor for 4 days. Relative cell growth was measured by counting with a hemacytometer. Figure S8. EMT primary breast cancer cells are more sensitive to ER stressors BT5104 (non-EMT) and BT5094 (EMT) cells were treated with solvent control, 0.1 µg/ml Tunicamycin (TM), or 10 nM Thapsigargin (TG) for 3 days. Cells were fixed with 70% ethanol, treated with RNase-A, and stained with propidium iodide. Fraction of cells in sub-G1 phrase was analyzed by FACS. Table S1 GSEA of microarray data from Cmp302-treated cells (A) Top 50 genes induced by Cmp302 in HMLE_Twist cells. UPR genes were highlighted. (B) List of genes in gene sets TM, TG, DOX and HYPOXIA Table S2 ECM gene expression in luminal and basal-B breast cancer lines 6 .
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