TGF-β1 was obtained from R&D systems (Minneapolis, MN). BAY11-7082 was purchased from Calbiochem (La Jolla, CA). The pcDNA3-BRMS1 plasmid was generated by subcloning EcoRI/XhoI from pOTB7-BRMS1 (clone ID: hMU011011, KRIBB, Korea). The empty pcDNA3 vector was used as a negative control.
All breast cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). MDA-MB-231 were maintained in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin. All cells were incubated at 37 °C under 5 % CO2 in a humidified incubator.
Small interfering RNA (siRNA)
siRNAs of HIF-1α was obtained from Sigma-Aldrich (St. Louis, MO). Control scrambled-siRNA was obtained from Invitrogen (Carlsbad, CA). Transfection was performed by utilizing Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Total cellular RNA was isolated from cultured cells using Trizol (Invitrogen, Carlsbad, CA), and 1 μg of RNA was reverse transcribed using oligo (dT) and M-MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s protocol. Reactions were performed as described previously . The primer sequences of mutants are shown below. HIF-1α; forward 5’-GTT TAC TAA AGG ACA AGT CAC C-3’ and reverse 5’-TCC TGT TTG TTG AAG GGA G-3’, TWIST1; forward 5’-GTC CGC AGT CTT ACG AGG AG-3’ and reverse 5’-CCA GCT TGA GGG TCT GAA TC-3’, E-cadherin; forward 5’-ACA GCC CCG CCT TAT GAT T-3’ and reverse 5’-TCG GAA CCG CTT CCT TCA-3’, Snail; forward 5’-TTT ACC TTC CAG CAG CCC TA-3’ and reverse 5’-GGA CAG AGT CCC AGA TGA GC-3’, Slug; forward 5’-TCT GCA GAC CCA TTC TGA TG-3’ and reverse 5’-AGC AGC CAG ATT CCT CAT GT-3’, GAPDH; forward 5’-ACA GTC AGC CGC ATC TTC TT-3’ and reverse 5’-ACG ACC AAA TCC GTT GAC TC-3’.
The cell lysates were prepared as described . Antibodies for HIF-1α (1:1000), p-p65 (1:1000), Snail (1:1000) and Slug (1:1000) were from Cell Signaling Technology (Danvers, MA). Antibodies for E-cadherin (1:1000), TWIST1 (1:1000), BRMS1 (1:500), p52 (1:500), p50 (1:1000) and GAPDH (1:3000) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibody for p65 (1:1000) was obtained from BD Biosciences (San Jose, CA). Secondary antibodies for anti-rabbit (1:2000 ~ 5000) and anti-mouse (1:2000) were Thermo Fisher Scientific Inc (Rockford, IL). Secondary antibody for anti-goat (1:3000) obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The immunoreactive bands were visualized by ECL (Thermo Fisher Scientific Inc., Rockford, IL) using ImageQuant 300 (GE Healthcare, Buckinghamshire, UK).
A TWIST1 luciferase reporter vector  was kindly provided from Dr. Hung, MC (M.D. Anderson Cancer Center, Houston, TX). A Snail luciferase reporter vector  was kindly provided from Dr. Yook, JI (Yonsei University College of Medicine, Korea). A HIF-1α luciferase reporter vector was obtained from Addgene (Cambridge, MA). The cells were prepared in 12-well plates in triplicate and transfected with the indicated reporter plasmids by utilizing Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). After stimulation with or without TGF-β1, the cells were washed twice with ice-cold PBS and harvested with a reporter lysis buffer (Promega, Madison, WI). The luciferase activity was analyzed as described previously .
In vitro invasion assay
In vitro invasion assay was performed with invasion assay kit with Matrigel-coated inserts (BD Biosciences, San Jose, CA) as described previously . Volume of 1 x 106 cells/well was added to the upper compartment of the invasion chamber. To the lower compartment, we added serum-free conditioned medium with or without TGF-β1. After incubation for 12 h (MDA-MB-231) or 48 h (MCF-7) at 37 °C, filters were fixed and stained with Diff-Quik reagents (Dade Behring, Inc., Newark, DE). The average numbers of six random microscopic fields (x200) was recorded in each experiment.
Immunofluorescence assays were performed as described previously . Antibodies for E-cadherin (1:100) and HIF-1α (1:50) were obtained from BD Biosciences (San Jose, CA). The cells were examined by confocal microscopy (LSM710; Carl Zeiss, Jena, Germany).
The ChIP assay was performed as described in the protocol from the Millipore ChIP Assay Kit (Upstate Biotechnology, Charlottesville, VA). Antibody for HIF-1α was obtained from Abcam (Cambridge, MA). The promoter-specific primers used were: TWIST1-HRE; forward 5’-GGA CTG GAA AGC GGA AAC TT-3’ and reverse 5’-CGA GGT GTC TGG GAG TTG G-3’, Snail-HRE; forward 5’-GCT GGG CCA GGC TGC TTT GCA-3’ and reverse 5’-GGA CAC CTG ACC TTC CGA CG-3’.
The cells were fractionated using the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. The fractionated samples were analyzed by Immunoblotting.
Data are shown as means ± s.d. Differences between two groups were assessed using the Student’s t-test. Differences among three or more groups were evaluated by analysis of variance, followed by Bonferroni multiple comparison tests.