Fig 2. Self-renewal of mammospheres derived from MCF7.
(A) The Sphere Formation Efficiency (SFE) was determined in MCF7-Tet-On-SrcDN. Thus, single cell suspensions from adherent cells were plated in 6-well ultralow attachment plates at 2x103 cells/well and cultured in mammosphere medium. Fifteen days later, mammospheres were dissociated into single cells that were divided into two groups: Control (-Doxy) and Doxy treated (2 μg/mL). Doxy-treatment was maintained for three generations, and renewed every 3 days. SFE at 3rd generation was calculated as number of spheres formed per number of seeded cells and expressed as % means ± SD. The SFE experiments were repeated 5 times, each one of them was carried out in triplicates. (p<0.05*). (B) Total cell extracts from the 3rd generation of mammospheres treated as above (panel A) were used to determine by immunoblotting expression of cyclin D1, PARP, with α-Tubulin, as a loading control, and SrcDN, Nanog, Oct3/4, ALDH1, and CD44, employing GAPDH or β-Actin, as a loading control. The net quantification of the gel bands after subtracting the background was carried out with ImageJ program and expressed in arbitrary units. These are representative results from 3 independent experiments. The ratios of the proteins SrcDN, Nanog, Oct3/4, ALDH1, and CD44 and their loading controls GAPDH or ß-Actin, or pY10-LDHA/LDHA were calculated and referred to -Doxy (Control) considered as 1.