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Additional Supporting Information may be found in the online version of this article.

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STEM_1061_sm_SuppFig1.tif1098KFigure S1. A subpopulation of Label-Retaining-Cancer-Cells (LRCC) is not quiescent and undergoes active cell-division. (A) Experimental procedure for the isolation of live LRCC and non-LRCC. (B) A FACS diagram shows Cy5+ LRCC (green) were successfully sorted out after 15 cell generations of growth in log phase post initial Cy5-dUTP labeling and sorting. (C-D) FACS analysis of Ki67+ cells show that a subpopulation of LRCC isolated from human cancer cell line and fresh primary human cancer cells are proliferating with 89.4% ± 3.3 of Ki67+ cells comparing to 79.2% ± 5.2 for non-LRCC (p=0.20). (E-F) FACS analysis of pHH3+ cells show that a subpopulation of LRCC isolated from human cancer cell line and fresh primary human cancer cells are actively dividing with 13.5% ± 2.5 of pHH3+ cells comparing to 6.5% ± 1.6 for non-LRCC (p=0.078). (G-I) Cell cycle analysis show that a subpopulation of LRCC isolated from human cancer cell line and fresh primary human cancer cells undergo active cell division with 16.9% ± 3.4 of G2/M phase cells comparing to 11.6% ± 2.7 for non-LRCC (p=0.28).
STEM_1061_sm_SuppFig2.tif2471KFigure S2. Cy5-dUTP labels DNA within nuclei in the time lapse confocal imaging experiments for real time detection of ACD-NRCC of live LRCC. (A) Experimental procedure of real time detection of live LRCC undergoing ACD-NRCC by DNA labeling with fluorescent Cy5-dUTP and time lapse confocal microscopy imaging. (B-C) Confocal images show that Cy5-dUTP co-localizes with DAPI staining supporting Cy5- dUTP labeling of DNA within nuclei. (D-E) Three dimensional confocal images re-constructed from serial single confocal images show that Cy5-dUTP labels DNA within nuclei.
STEM_1061_sm_SuppInfo.pdf106KSupplementary Data
STEM_1061_sm_SuppTab1.pdf21KTable S1. Determination of cell doubling times. To effectively detect asymmetric cell division, we determined the growth curves and doubling times experimentally for all cell lines and the fresh tested tumor cells. Growth curve's correlation value R2≥0.9 was considered adequate for computations of doubling times.
STEM_1061_sm_SuppVideo1.avi5624KMovie S1. A time-laspe confocal microscopy movie showing LRCC undergoing ACD-NRCC in live cells and in real-time.

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