CSL-independent Notch signaling pathway might play a role in liver CSCs and MRK003 could partly eliminate the stem-like cells. (A) The downstream proteins levels of the CSL-independent Notch pathway (DTX1 and Ep300) were significantly increased in the PLC/PRF/5 spheres, compared with the parental cell line. GAPDH was used as a loading control. (B) 10 μM MRK003 could reduce the activation of Notch1 and downstream components of CSL-independent Notch signaling pathway DTX1 and Ep300 were consequently decreased. (C) The secondary dissociated PLC/PRF/5 sphere-forming cells were treated with 10 μM MRK003 or DMSO control for 7 days. The sphere formation ability of the MRK003-treated groups were significantly inhibited in comparison to DMSO-treated controls.