Additional file 2: Figure S2. of Identification of EGF-NF-κB-FOXC1 signaling axis in basal-like breast cancer

Inhibition of NF-κB p65 affects FOXC1 protein levels. MDA-MB-468 cells were serum-starved overnight and treated with EGF for 24 h after pre-incubation with NF-κB inhibitors, Bay 11–7082 or BMS-345541, for 1 h. FOXC1 protein levels were examined by immunoblotting. (TIFF 56 kb

Additional file 1: Figure S1. of Identification of EGF-NF-κB-FOXC1 signaling axis in basal-like breast cancer

NF-κB transcription factor mediates EGF-induced FOXC1 expression in multiple breast cancer cell lines. a MDA-MB-231 and BT-20 cell lines 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 < 0.05; **, P < 0.001. b MDA-MB-468 cells were transiently transfected with the IKKβ or SR-IκBα constructs and immunoblotted for FOXC1 expression. c MDA-MB-231 and BT-20 cell lines were treated with 100 ng/mL EGF for 2 h after preincubation with the NF-κB inhibitor Bay 11–7082 for 1 h. FOXC1 mRNA levels were examined using qRT-PCR. **, P < 0.001. d Total protein was extracted from MDA-MB-468, MDA-MB-231 and BT-20 cell lines after no starvation or treatment (Left to right, first three lanes) or after serum-starvation overnight with or without EGF treatment for 24 h. EGFR and phosphorylated EGFR (p-EGFR) levels were examined with immunoblotting. e BT-20 cells were treated with 100 ng/mL EGF for 1 h after preincubation with the Akt inhibitor-IV or U0126 (ERK inhibitor) for 45 min. Total protein was extracted and phospho-p65 (Ser 536) was examined by immunoblotting. (TIFF 131 kb