Fig. 4

Arrb1 mediates acetylation of Gli1. a Arrb1 and Gli1 protein levels in CSCs transfected with the arrb1-HA plasmid and analyzed 48 h after transfection. LC: Actin. b WB analysis of endogenous Arrb1, Gli1 and its acetylated form (Gli1-ac) in CSCs cultured in SM in DFM for the indicated time points. LC: Actin. c Arrb1, Gli1 its acetylated form (Gli1-ac) protein levels in CSCs transfected with the arrb1-HA plasmid and analyzed 8 h after transfection. LC: Actin. d Arrb1, acetylated Gli1 (Gli1-ac) and p300 protein levels in CSCs transfected with control siRNA (siCtr) or Arrb1 siRNA (siArrb1) cultured for 12 h in DFM. LC: Actin. e HEK293T cells were transfected with the Arrb1-HA, Gli1-Flag and p300-HA plasmids, alone or in combination. Left panel: whole cell extracts were immunoprecipitated with anti-Flag agarose beads and immunoblotted with anti-HA, anti-Flag and anti-Gli1-ac antibodies. As negative controls, beads were pre-blocked with excess Flag peptide (0.1 mg/ml). Right panel: 2,5% of the immunoprecipitated cell lysates (input) were immunoblotted with the indicated antibodies. These experiments show that Arrb1 forms a complex with p300 and Gli1. f Luciferase activity of Gli-responsive reporter (Gli8x_luc) in CSCs co-transfected with a wild-type Gli1-flag expression vector (Gli1 wt) or a Gli1K/R mutant (Gli1 K518R) together with the arrb1-HA or an empty vector as control. *p < 0.05 vs Empty; **p < 0.05 vs Gli1 wt. g Left panel: Oncosphere forming assay in CSCs after ectopic expression of Arrb1. Data represent mean ± S.D. from three independent experiments. *p < 0.05. Right panel: WB analysis of endogenous Nanog along with ectopic Arrb1 in CSCs overexpressing Arrb1 or empty vector as control (Ctr). LC: actin. h BrdU uptake in CSCs after ectopic expression of Arrb1. Data represent mean ± S.D. from three independent experiments. *p < 0.05