Figure 3.

Calpain affects mammosphere forming efficiency. Control and CAPNS1 depleted MCF7 and MCF10AT cells were plated in 96 wells multiwell plates (1000 cells/well) in mammosphere specific medium. After ten days, the mammospheres were collected and disaggregated. Mammospheres derived cells were counted and plated as for the first generation and 7 d later picture were taken. (A) typical MCF7 mammospheres. Scale bar corresponds to 50 μm. (B) reports the average number of second generation MCF7 mammospheres obtained in 3 independent experiments (p values <0.01). (C) typical MCF10AT mammospheres. Scale bar corresponds to 50 μm. (D) reports the average number of second generation MCF10AT mammospheres obtained in 3 independent experiments (p values <0.01). (E) shows the average number of mammospheres counted in 3 independent experiments in control and shCAPNS1 MCF10AT treated or not treated with thapsigargin (p values<005). Briefly, control and CAPNS1 depleted MCF10AT cells were plated on 96 wells multiwells (1000 cells/well) in mammosphere specific medium. After 7 days, the mammospheres were incubated or not with 100 nM thapsigargin for 6 hours. Mammospheres derived cells were counted and plated as for the first generation and 7 d later the number of second generation mammospheres was counted. Control and CAPNS1 depleted MCF10AT cells derived from second generation mammospheres were stained with anti CD44 and anti CD24 antibodies and analyzed by FACS. Adherent cells were analyzed in parallel. The dot plot (F) and the graph (G) are the results of a typical FACS analysis.