20(S)-Hydroxycholesterol (20S) and Shh induce Notch target genes independent of the canonical Notch signaling pathway. (A) M2 cells at 70% confluence in a 24 well plate were transiently transfected with CBF-1 luciferase reporter construct pTK-luciferase plasmid and pTK-Renilla-luciferase plasmid (Promega, Madison, WI, USA) using Fugene 6 Transfection Reagents from Roche (Indianapolis, IN, USA). Twenty-four hours after transfection, the cells were treated with control vehicle, Notch interacellular domain (NICD) overexpression vector, 5 µM 20S, or 200 ng/mL Shh for 24 and 48 hours. Notch activation of CBF-1 was normalized to Renilla luciferase activity. Transfection efficiency was monitored by cotransfecting with a plasmid expressing green fluorescent protein. (B, C, E, F) M2 cells were treated at confluence with control vehicle or 5 µM 20S or cultured on 2.5 or 5 µg/mL immobilized Jagged-1 for real-time PCR and alkaline phosphatase (ALP) activity analyses. After 48 hours of treatment, HES-1, HEY-1, ALP, and bone sialoprotein (BSP) mRNA expression was measured by quantitative real-time PCR. (D) After 72 hours of treatment, ALP activity using whole-cell extracts was measured by a colorimetric method. Fold changes in gene expression compared with the control cells were calculated using the ΔΔCt method and reported as the mean of triplicate determination ± SD. (G) M2 cells at confluence were treated with control vehicle (control), cultured on 5 µg/mL immobilized Jagged-1, or treated with 5 µM 20S. After 48 and 72 hours of treatment, nuclear extracts were collected for Western blotting using antibodies to NICD and β-actin. (A) ***p < .0001 for control versus NICD. (B) ***p < .0001 for control versus 20S and Jagged-1 (2.5 and 5 µg/mL). (C) **p < .001 for control versus 20S; ***p < .0001 for control versus Jagged-1 (2.5 and 5 µg/mL). (D–F) ***p < 0.0001 for control versus 20S.