Hes1 upregulated MMP-9 through activation of NF-κB signaling pathway. (A) The full-length MMP-9 (−1284/+21) (upper panel), a truncation mutant (−584/+21) (middle panel), or the full-length MMP-9 with mutations of SP-1– and AP-1–binding sites (lower panel) inserted into pGL3 basic expression vector are shown as pGL3-MMP-9-Luc, pGL3-truncated MMP-9-Luc, or pGL3-mutated MMP-9-Luc, respectively. Binding sites for the 3 transcription factors (NF-κB, SP-1, and AP-1) are shown (left panel). 293T cells were transiently transfected with either pMYs-Hes1-IG or pMYs-IG together with pGL3-MMP-9-Luc (upper panel), pGL3-truncated MMP-9-Luc (middle panel), or pGL3-mutated MMP-9-Luc (lower panel). (B) 293T cells were transiently transfected with the indicated expression plasmids (pMYs-Hes1-IG, pMYs-IG, pMX-SR-IκBα-IRES-Zeocin, pMX-IRES-Zecoin) together with pGL3-NF-κB-Luc (left panel). 293T cells were transiently transfected with either pMX-SR-IκBα-IRES-Zeocin or pMX-IRES-Zecoin together with pGL3-NF-κB-Luc. After 36 hours, transfected cells were cultured in the presence or absence of 20 ng/mL TNFα for 12 hours (right panel). (C) 293T cells were transiently transfected with pMX-SR-IκBα-IRES-Zeocin and either pMYs-Hes1-IG or pMYs-IG together with pGL3-MMP-9-Luc. (A-C) Results represent the average values for relative luciferase activity. All transfection groups were normalized with a Renilla luciferase vector as an internal control. (D) ELISA assay for NF-κB subunits in Hes1-ER– or mock-transduced M1 cells cultured in the presence or absence of 4-hydroxy-tamoxifen (4-OHT) for 48 hours. (A-D) All data points correspond to the mean and SD. Data are representative of 3 independent experiments. Statistically significant differences are shown: *P < .05, **P < .01.