Fig 2: Flow cytometric analysis of ADSCs. Cell surface antigens of the ADSCs were similar to those of bone marrow mesenchymal stem cells. The expression of (A) control, (B) CD29, (C) CD44, (D) CD71 and (E) CD73 was detected by flow cytometry. The results indicated that ADSCs at P5 exhibited high expression of CD29, CD44, CD71 and CD73. ADSCs, adipose-derived stem cells; P5, passage 5.

Fig 3: Surface markers of the ADSCs. Surface markers of the ADSCs were similar to those of bone marrow mesenchymal stem cells. The expression of CD29, CD31, CD44, CD71 and CD73 was detected by RT-PCR and immunofluorescence. (A) RT-PCR analysis indicated that the ADSCs expressed CD29, CD44, CD71 and CD73, but not CD31. GADPH was used as an internal control. (B) Immunofluorescence demonstrated immunopositivity for CD29, CD44, CD71 and CD73 (scale bars, 50 µm). ADSCs, adipose-derived stem cells; RT-PCR, reverse-transcription polymerase chain reaction analysis; P3, passage 3.

Fig 4: Isolation and identification of ADSCs. (A) The morphology of ADSCs is presented. Scale bar, 50 µm. (B) The ADSCs were identified by the positive and negative markers, including CD90, CD44, CD34, and CD45, using immunofluorescence analysis. Scale bar, 50 µm. (C and D) The GFP-labeled ADSCs was identified by in vivo fluorescence tracing. Scale bar, 50 µm. ADSCs, adipose-derived mesenchymal stem cells.

Fig 5: Deprivation of NO elevates self-renewal potential of non-malignant mammary epithelial cells. (A) Representative images of mammospheres derived from MCF10A cells grown on PolyHEMA-coated plates under the treatment of control (PBS), L-arginine or L-NAME for one week. Scale bars: 200 µm (top 2 rows) and 50 µm (bottom row). (B) The size of mammospheres derived from drug-treated MCF10A cells. (C) The number of spheroids formed at each seeding number. (See the micrographs of spheroids in Supplementary Fig. 7A). (D) Mammosphere forming efficiency at each seeding number. Mammosphere forming efficiency (%) = (# of spheroids)/(# of cells seeded) × 100119,120. Statistical significance between different treatments was determined by two-way ANOVA analysis with Bonferroni post hoc test175. Error bars: mean ± STDEV. **p < 0.01; ***p < 0.001. (E) Staining of mammospheres for the basal marker, CK14, and luminal marker, CK8/18. Co-localization of CK14 and CK8/18 was captured with ImageJ. (See the quantification in Supplementary Fig. 7B). (F) Limiting dilution analysis to determine the sphere forming frequency at clonal densities (=200 cells/96-well)126. (Top) The graph plotted with the Y-axis as Ln (fraction of negative wells) and X-axis as seeding number. (Bottom) The sphere forming frequency corresponding to -(slope) of the line of best fit. (G) Staining of paraffin-embedded/sectioned mammospheres129 for stem cell markers CD44 and CD24. Scale bars: 20 µm (see the quantification of CD44 and CD24 levels in Supplementary Fig. 7C,D). (H) FACS analysis to determine the expression of CD44 and CD24 in cells dissociated from mammospheres. Isotype controls were used to set threshold gate.