Figure 2.

CR-CSCs Differentiation Is Associated With sEVs Release. (a) Treatment with 20 mmol/L NH₄Cl or with 10 μmol/L GW4869, which block multivesicular body maturation, inhibits differentiation of CR-CSCs Sa41 and Re121, as assessed by evaluating ALP activity (Vmax) and (b) prevents the reduction of the percentage of CD133/CD44 positive cells (from 19% to 60% and 42% for NH₄Cl and GW4869, respectively) but similar results were obtained with the other CR-CSC lines tested in this study (Figure S3). CSC = cancer stem cells undifferentiated; FBS = CR-CSCs differentiated with 10% FBS and in absence of growth factors; NaB = CR-CSCs differentiated with 10% FBS, maintained in absence of growth factors and treated after seven days with 2 mM NaB for 48 h. NaB NH₄Cl = CR-CSCs differentiated with 10% FBS, maintained in absence of growth factors and treated after seven days with 2 mM NaB and 10 mM of NH₄Cl for 48 h; NaB GW4869 = CR-CSCs differentiated with 10% FBS, maintained in absence of growth factors and treated after seven days with 2mM NaB and 10 μM of GW4869 for 48 h. *, p ≤ 0.05; ****, p ≤ 0.0001.