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Figure S1. Frequent observation of CD44+CD24neg/lo surface expression in ER negative breast cancer lines and tumour cultures. (A) ER negative breast cancer cell lines. (B) TNBC-derived dissociated tumour (DT) cultures. (C) ER positive lines and (D) equal numbers of mixed MDA-MB-231 and MCF7 cells.

Figure S2. ESA expression and aldefluor activity are observed in subsets of CD44+CD24low+ cells. (A) CD44 and CD24 in DT-25 at passage (P3) compared to DT-25 at passage (P9). (B) Serial mammospheres formed from sorted CD44+CD24low+ and CD44+CD24neg from DT-25. Mean ± SEM, *p = 0.0054. (C) Aldefluor activity (top) and flow cytometry for surface ESA (bottom) were assayed as described in MDA-MB-231 and DT-22. Respective unstained controls are shown. (D) Cells were gated for ESA+ (left) or ALDH1+ (right) and CD44/CD24 assayed.

Figure S3. CD44+CD24low+ give rise to both CD44+CD24low+ and CD44+CD24neg progeny while CD44+CD24neg yield only CD44+CD24neg. CD44+CD24low+ or CD44+CD24neg were sorted from DT-22 and DT25 and 100,000 cells cultured. (A and D) Population growth from sorted CD44+CD24low+ and CD44+CD24neg. (B and E.) The proportion of CD44+CD24low+ or CD44+CD24neg cells arising from CD44+CD24low+ cells is shown over time. (C and F) Growth curves of progeny of CD44+CD24low+ over 14 days. As for MDA-MB-231 (Fig 3A) CD44+CD24neg generated only CD44+CD24neg cells over 14 days (not shown here).

Figure S4. Expression of lung, brain and bone metastasis gene expression signatures is enriched in CD44+CD24low+ subpopulations from MDA-MB-231 and DT-22. (A–C) Expression of lung (A), brain (B) and bone (C) metastasis gene expression signatures is enriched in CD44+CD24low+ subpopulations in MDA-MB-231. Gene set analysis (GSA) compared enrichment of indicated signatures. Shown are ordered gene scores for each gene in the line plot and the average fold change in the heatmap (orange indicates high expression in CD44+CD24low+ and blue is low). Average fold gene expression changes are indicated by bar graphs. Upper left panel shows enrichment score and the p-value. (D and E) Expression of lung (D) and brain (E) metastasis gene expression signatures is enriched in CD44+CD24low+ subpopulations in DT-22 analysis as above. A list of the genes used for each signature and their individual gene scores are in shown in Supporting Information Table S1.

Figure S5. Expression of embryonic stem cell transcription factors and targets is enriched in CD44+CD24low+ subpopulations. (A–F) GSA comparing enrichment of indicated embryonic stem cell transcription factors (A, B, D and E) and genes upregulated by NOS (Nanog, Oct4 and Sox2). (C and F). The “NOS TF up” is a gene set of transcriptional regulators identified in human ES that overexpress Nanog, Oct4 or Sox2. The “NOS Targets up” gene set contains the activated genes from the ChIP-array for Nanog, Oct4 and Sox2. A list of the genes used for each signature and their individual gene scores are in shown in Supporting Information Table S1. For MDA-MB-231 (A–C) and DT-22 (D–F) the ordered scores for each gene in the line plot and the average fold change in the heatmap are shown (orange indicates high expression in CD44+CD24low+ and blue, low). Enrichment scores and p values are at upper left. Average fold gene expression changes are shown in bar graphs and a list of genes and their individual gene scores are in Supporting Information Table S1.

Figure S6. GSI sensitivity is restricted to the CD44+CD24low+. (A–E) CD44+CD24low+ (CD24low+) and CD44+CD24neg (CD24neg) cells sorted from MDA-MB-231 (A and B), DT-22 (C and D) and DT-25 (E) were treated with or without DAPT. Effect of DAPT on cleaved Notch1 (N1-ICD) and Sox2 ± after 24 h of 5 and 10 µM DAPT in CD44+CD24low+ cells (A and C). Serial mammospheres of indicated cells from sorted populations ±5 μM DAPT (mean ± SEM, Student's t-test). The p values in panel B: *p = 0.00001; **p = 0.00002; ***p = 0.0001; panel D: *p = 0.0045; **p = 0.006; ***p = 0.0054; panel 10. (E) *p = 0.0027; **p = 0.0005; ***p = 0.007 Serial mammospheres from DAPT-treated CD24low+ cells were significantly reduced compared to respective untreated controls. *Statistically different by student's t-test from control primary spheres. Treated and untreated tertiary mammospheres from CD24neg cells were reduced significantly compared to primary spheres, but both were unaffected by DAPT (B, D and E). (F) Mean soft agar colonies arising from sorted populations of MDA-MB-231 ±5 μM DAPT (mean ± SEM, t-test, *p = 0.04). (G) Matrigel invasion of sorted populations from DT-22 ±10 µM RO4929097 (RO) generated by xCELLigence real time cell analysis, graphed as mean ± SEM. CD24low+ cells show significantly greater invasion compared to CD24neg cells (**p = 0.0055 at T = 12 hrs, Student's t-test). RO significantly attenuated invasion (*p = 0.004 at T = 12 h, Student's t-test) of CD24low+ cells. CD24neg cells were unaffected. (H) Cell cycle profiles of sorted populations from MDA-MB-231 after 48 h ±10 µM RO4929097. (I and J) GSI does not affect MDA-MB-231 or DT-22 cell proliferation. Proliferation curves of 100,000 cells from sorted CD44+CD24low+ or CD44+CD24neg populations from MDA-MB-231 (I) and DT22 (J) cultured over 12 days ±10 µM RO4929097.

Table S1. Individual gene scores from GSA results showing enrichment of various gene signatures in sorted CD44+CD24low+ compared to CD44+CD24neg subpopulations from MDA-MB-231 and DT-22 cells. Higher gene scores indicate increased expression while lower gene scores indicate decreased expression.

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