Fig 2: Constitutive Myc Expression Increases the Rate of MaSC Symmetric Divisions(A) Representative confocal images of asymmetric and symmetric divisions in, respectively, control (Ctrl) and 4-OHT-treated (MycER) Rosa26-MycER cells. Cells were stained with DAPI, aMyc, and aCD49f antibodies. Scale bar, 10 µm.(B) Percentage of asymmetric and symmetric divisions in Ctrl and 4-OHT-treated (MycER) Rosa26-MycER cells based on the distribution of the CD49f marker. *chi-square p < 0.0001.(C) Experimental scheme of the PKH26 label-retaining assay.(D) Percentage of asymmetric and symmetric divisions in LTR-Ctrl and LTR-MycER PKHhigh cells (left) and R-Ctrl and R-MycER bulk mammospheres (right), based on Numb distribution. *chi square p < 0.0001.(E) Immunofluorescence staining for DAPI, Myc, and Numb expression in 4-OHT-treated (200 nM) WT Ctrl (Ctrl) and Rosa26-MycER (MycER) doublets. Representative images depict an asymmetric (top) and a symmetric (bottom) division. Scale bar, 10 µm.

Fig 3: Infant gastric digestion kills all cell types equally, except for immune cells, which are less resilient to digestion. A) Cell populations were identified using antibody staining and subsequent flow cytometry analysis. The relative percentages of defined viable cell populations in milk before and after digestion did not change, with the exception of immune cells, which did not survive digestion. (n = 6 replicates from milk expressed on different days). B) The ALDEFLUOR assay measured stem cell populations before and after simulated digestion. High ALDH signal indicates stem-like phenotypes. ALDEFLUOR staining found no change in stemness following digestion. DEAB is an inhibitor of ALDH and was used to control for background fluorescence (n = 5). C) Digested and undigested milk cells were analyzed by flow cytometry for the presence of stem and progenitor cell markers NANOG, SOX2, CD49f, and TRA-1-60. No changes in stemness were detected following digestion (n = 5). D) Digested and undigested milk cells were analyzed by qPCR for the presence of stem cell markers. No significant changes were detected (n = 5). In all panels, significance was determined by Student's t test compared to undigested control cells, with *p < 0.05. Error bars represent standard deviation.