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sc-12-0042_sm_SupplFigure1.pdf1001KSupplemental Figure 1. Direct immunofluorescence analysis of surface ABCG2 expression. Surface membrane ABCG2 expression was determined by directly immunostaining with PE-labeled anti-human CD338 (ABCG2) (clone 5D3) in (A) a differentiated TE03 colony that overexpressed ABCG2, (B) undifferentiated TE06 cells (TE06 undif), (C) spontaneously differentiated TE06 cells (TE06 Dif), (D) and mixed populations of TE06 cells that contain morphologically differentiated cells with a negative ABCG2 status as indicated by arrowheads. The ABCG2+ cells were found in morphologically undifferentiated cells as indicated by arrows. Both TE03 and TE06 cells were grown on 2.5% Matrigel in mTeSRTM1 medium. Under this growth condition, there were approximately 5 to 8% of spontaneously differentiated cells observed in TE06 cells (C), whereas the TE03 line had approximately 2% of spontaneously differentiated cells (A). The differentiated colonies, based on morphologies and other immunostaining criteria, were counted under microscope. The images were acquired under an Axiovert 200 fluorescence microscope (Zeiss). Bar, 50 μm
sc-12-0042_sm_SupplFigure2.pdf1145KSupplemental Figure 2. (A): Analysis of surface ABCG2, SSEA-1, and SSEA-4 coexpression during BMP-4 directed differentiation. Surface ABCG2, SSEA-1, and SSEA-1 were co-stained with anti- SSEA-1, anti-SSEA-4, and anti-ABCG2 (clone 5D3) in BG03 control cells and BG03 cells treated with 50 ng/ml or 100 ng/ml of recombinant BMP-4 for 4 days (d4). (B-C): Immunofluorescence analysis of surface ABCG2, SSEA-1, and SSEA-4 coexpression under cytotoxic stresses. Surface membrane coexpression of ABCG2, SSEA-1, and SSEA-1 was determined by immunostaining the cells with anti- ABCG2 (clone 5D3) in (B) BG03 control, (C) BG03 cells treated with 1 μM mitoxantrone for 24 hour, and (D) BG01 cells treated with 10 nM doxorubicin for 24 hour. Both BG01 and BG03 cell lines were cultured on 2.5% BD Matrigel in mTeSRTM1 medium. Less than 1% of the colonies were observed to have spontaneously differentiated cells under these culture conditions. Homogeneously staining patterns were observed in all experimental panels except in Supplementary Figure 2C, in which only one colony survived and was photographed from one well of a 6-well plate. All other previously seeded colonies (∼ 100 colonies) were killed by the mitoxantrone (MX) treatment. The images were acquired with an Axiovert 200 fluorescence microscope (Zeiss). Bar, 50 μm
sc-12-0042_sm_SupplFigure3.pdf2496KSupplemental Figure 3. Surface ABCG2 expression in BG03 cells. Surface membrane ABCG2 expression was determined by direct immunostaining of the cells with PE-labeled anti-human CD338 (ABCG2) (clone 5D3): (A) undifferentiated BG03 cells; (B) spontaneously differentiated BG03 cells, (C) morphologically undifferentiated BG03 colonies (2 out of ∼ 100 colonies) stained with the 5D3 monoclonal antibody; (D) Enlarged view of C; (E) Two 5D3 positive colonies shown in D were isolated by a colony pick-up method under a Leica microscope, triturated into small clumps, and plated on a new well coated 2.5% BD Matrigel. All clumps formed nice colonies after the immediate passage, without recurrent 5D3 staining and spontaneous differentiation. Bar, 50 μm

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