Fig. 1
From: Identification of EGF-NF-κB-FOXC1 signaling axis in basal-like breast cancer
NF-κB transcription factor mediates EGF-induced FOXC1 expression. a MDA-MB-468 cells were transiently co-transfected with the FOXC1 promoter-luc and NF-κB (p65), IκBα S32A/S36A super-repressor (p65 + SR-IκBα), or the vector. Reporter activities were assessed by luciferase assays. ***, P < 0.0001. b MDA-MB-468 cells were transiently transfected with the FOXC1 promoter-luc and IKKβ construct or treated with 100 ng/mL EGF for 24 h, followed by luciferase assays. ***, P < 0.0001. Data represent mean ± SD from 3 independent experiments. c Left, MDA-MB-468 cells were transfected with p65 siRNA for 48 h and then treated with or without EGF for 6 h. FOXC1 mRNA levels were examined using qRT-PCR. n.s., not significant; **, P < 0.001; *, P < 0.05. Right, MDA-MB-468 cells were transfected with p65 siRNA for 48 h and then treated with or without EGF for another 24 h. FOXC1 and p65 in nuclear and cytoplasmic fractions were examined using immunoblotting. Lamin A/C was used as a nuclear marker and actin was used as a cytoplasmic marker. d WT, p65-null, and p65-reconstituted MEFs were transfected with the FOXC1 promoter-luc and immunoblotted for p65 protein expression and then (e) , treated with EGF or vehicle for 24 h, followed by luciferase assays. **, P < 0.001; ***, P < 0.0001. f WT or p65−/− MEFs were co-transfected with the FOXC1 promoter-luc and pBABE-EGFR construct, followed by luciferase assays. **, P < 0.001. Data represent mean ± SD of 3 independent experiments. g MDA-MB-468 cells were transiently transfected with the NF-κB-luc construct. After 30 min pre-treatment with 5 μM U0126 (MEK inhibitor) or 1 μM AKTIV (AKT inhibitor), cells were stimulated with EGF for 24 h in the presence or absence of the inhibitors. Reporter activities were measured by luciferase assays. **, p < 0.01; ***, P < 0.0001. h Wild-type and p65−/− MEFs were co-transfected with the FOXC1 promoter reporter construct and constitutively active Akt1, Akt3 or ERK2 constructs for 24 h. FOXC1 promoter activity was assessed by luciferase assays. *, P < 0.05; **, P < 0.01; ***, p < 0.0001