Supplementary MaterialsAdditional file 1 Physique showing specificity for ALDH1A1 and ALDH1A3 antibodies used in immunostainings. ALDEC primary human mammary epithelial cells separated by with ACS were immunostained for ER (FITC) and reanalyzed with flow cytometry. ALDEC cells contained 27.8% ER+ cells CA-4948 (left panel), whereas ALDE+ cells did not express ER above background level (right panel, 3.8% of ALDE+ population, 0.002% of the total population). (B) Breast cancer cell lines SUM44 (ER+ cell line) and SUM149 (ERC cell line) were used as positive (left panel) and unfavorable (right panel) control for ER expression. The 3.8% positive cells detected with flow cytometry in the ALDE+ cell population (A) represent background staining, as indicated by the presence of 5.1% ER+ cells in SUM149 ERC breast cancer cells, which was similarly immunostained and similarly gated for flow-cytometry analysis. (C) Immunostaining for ER on ALDE-sorted cells showed ER+ cells in the ALDEC population, but not in the ALDE+ cell population. (D,E) Double staining for ALDH1A3 and ER on normal breast sections show no colocalization. (F-I) Double staining for ALDH1A1 and ER on normal breast sections showing representative areas with ERlow/ALDH1A1+ cells (arrows) in two different mammoplasty samples (H, I). ERhigh/ALDH1A1C cells in the same sections are indicated with arrowheads. (J) Quantitative assessment of ERlow/ALDH1A1+ cells in normal breast samples revealed a small percentage of double-positive cells only in three of 11 samples. Scale bar = 50 m. bcr3663-S3.pdf (3.7M) GUID:?693913E0-FA58-4941-8681-BE16928B5E25 Additional file 4 Figure showing strategy for identification and isolation of ER+ and ERC cells from normal mammary epithelium. (A, B) Diagram of experimental actions and reporter construct used for the separation of ER+ and ERC cells. (C) Level of ER expression as reported by level of GFP expression in MCF7 ER+ breast cancer cells, MDA-MB-231 ERC breast cancer cells and primary normal mammary epithelial cells (HMEC). (D) Immunostaining for ER expression on cytospins from GFP-sorted cells transduced with the Ade 25 ERE Pr GFP construct. Representative images of ERC cells (upper panel) and ER+ cells (lower panel) after separation with the reporter system. Nuclei were detected with PI staining. GFP+ cells contained 95% ER+ cells by immunostaining, and GFPC cells contained 2% ER+ cells. bcr3663-S4.pdf (554K) GUID:?EEA34E0E-8C9C-4083-91B9-46604FFE4F1C Additional file NDRG1 5 Figure showing double staining of mammospheres for ER and Ki67. Mammosphere sections were double stained for ER (green) and proliferation marker Ki67 (red). White arrows indicate double-positive cells. Scale bar = 25 m. bcr3663-S5.pdf (394K) GUID:?A60DBDB0-C4D4-47CB-9869-21AA95AD97BC Additional file 6 Table showing outgrowth potential of normal mammary epithelial cell subpopulations sorted for ER in the humanized fat pad of NOD/scid mice. bcr3663-S6.pdf (43K) GUID:?20A9CC9B-E594-4EA5-A3CC-FA71F546C093 Additional file 7 Figure showing primary and secondary mammosphere formation after shRNA knockdown of ALDH1A1. (A) Primary sphere formation after ALDH1A1 KD CA-4948 with two different shRNA constructs (9 and 10) as well as using a pool of these CA-4948 two shRNAs (9+10). (B) Primary and secondary sphere formation after ALDH1A1 KD with combined shRNAs #9 and #10. values given are compared with NT control and were calculated by using a two-tailed test. bcr3663-S7.pdf (67K) GUID:?4E6DF22E-D4E2-4168-AF7D-27D2741C293D Additional file 8 Figure showing RAR staining in normal breast. Nuclear RAR was expressed in the vast majority of breast epithelial and stromal cells, although occasional negative nuclei were detected in both epithelium and stroma (arrows). Scale bar = 100 m. bcr3663-S8.pdf (1.8M) GUID:?DD217394-D871-4ADE-A2A7-65ED22BB4300 Abstract Introduction Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ERC, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects.