Cell lines and culture conditions
The 21T parental cell lines (21PT, 21NT, and 21MT-1) were a kind gift from Dr. Vimla Band (Dana Farber Cancer Institute, Boston, MA) [18] and were cultured in alpha modification of Eagle’s medium supplemented with 2.8 μM hydrocortisone (H), 12.5 ng/ml epidermal growth factor (E), 2 mM L-glutamine, 1 μg/ml insulin, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 50 μg/ml gentamycin reagent (called αHE – all supplements from Wisent Bioproducts). For regular culture conditions, the αHE media was further supplemented with 10 % fetal bovine serum (FBS) (Sigma-Aldrich) and named αHE10F. Stably transfected cells were cultured in αHE10F containing either 500 μg/ml G418 (Wisent Bioproducts) or 0.8 μg/ml puromycin (Sigma-Aldrich).
Generation of TBX3 expression vectors
Expression vectors were constructed for each TBX3 isoform (TBX3iso1and TBX3iso2). To obtain TBX3iso2, PCR amplification of the whole transcript was performed from a pOTB7 expression vector containing TBX3iso2 [Genbank:BC025258] (Open Biosystems Thermo Scientific) as the cDNA template using Phusion High-Fidelity DNA Taq polymerase (New England BioLabs). Primers used to amplify TBX3iso2 were: forward: 5′-GCC ACC ATG AGC CTC TCC ATG AGA-3′ and reverse: 5′-TTC GGG ACC GCC TGC GGG ACC TGT CCG GC-3′. To produce TBX3iso1, two PCR product fragments, representing the transcript before (fragment 1) and after (fragment 2) the 66 bp TBX3iso2 addition, were PCR amplified. Primers used for fragment 1 of TBX3iso1 were: forward: 5′-GCC ACC ATG AGC CTC TCC ATG AGA-3′ and reverse: 5′-CAT GGA GTT CAA TAT AGT AAA TCC ATG TTT GAC-3′. Primers used for fragment 2 of TBX3iso1 were: forward: 5′-TGG ATT TAC TAT ATT GAA CTC CAT GCA CAA AT-3′ and reverse: 5′-TTC GGG ACC GCC TGC GGG ACC TGT CCG GC-3′. All primers used for generation of the TBX3 expression vectors were purchased from Sigma-Aldrich. Products were separated on 1 % agarose gel and bands representing TBX3iso2 at ~2000 kb were extracted and pooled. For TBX3iso1, a band at ~660 kb for fragment 1 and a band at ~1380 kb for fragment 2 were gel extracted and pooled. When designing the PCR primers for the two fragments of TBX3iso1, the reverse primer of the upstream amplicon (fragment 1) and the forward primer for the downstream amplicon (fragment 2) had a 20 bp overlap to ensure that the ends of these amplicons would anneal in a subsequent PCR reaction to melt the fragments together. This annealed TBX3iso1 PCR product was purified and amplified again. After running this PCR product on a 1 % agarose gel and extracting at ~2000 bp, both TBX3iso1 and TBX3iso2 PCR products were incubated with T4 polynucleotide kinase (New England BioLabs), and purified. Both products were inserted separately into pZsGreen-C1 plasmids (Clonetech Laboratories), such that the ZsGreen was fused to the C-terminus of TBX3. To prepare the pZsGreen-C1 plasmid, it was digested with Afe1 (New England BioLabs) and incubated with calf intestinal alkaline phosphatase (New England BioLabs) to dephosphorylate the cut ends. The digested plasmid was electrophoresed and gel extracted. The plasmid and TBX3 PCR products were incubated overnight with ATP and T4 DNA Ligase (New England BioLabs) at 16 °C to complete ligation. Competent bacteria (DH5alpha) were transformed with the ligated plasmid and kanamycin resistant clones were expanded to isolate DNA. Clones were digested with the following enzyme pairs to check for proper size and orientation: AgeI/KpnI, MfeI/XhoI, and NheI/HndIII. Suitable clones were sequenced (DNA Sequencing Facility at Robarts Research Institute, London, ON).
The use of untagged TBX3 was required so the TBX3 expression vectors underwent site directed mutagenesis in order to introduce a stop codon at the end of full length TBX3iso1 and TBX3iso2 using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s protocol. Clones were sent to the DNA Sequencing Facility at Robarts Research Institute for sequencing. Clones with the proper sequence were used for stable transfections.
Transfections
Purified TBX3iso1 and TBX3iso2 plasmid DNA was transfected using PolyJet DNA In Vitro Transfection Reagent (SignaGen Laboratories) following the manufacturer’s protocol. Empty vector (EV) plasmids were transfected as a control. Stable transfectants were selected in αHE10F containing 500 μg/ml G418 (Wisent Bioproducts). Approximately two weeks post-transfection, resistant clones were pooled, expanded, and frozen for later use.
Generation of lentiviral particles and transduction
Generation of short hairpin (sh) RNA containing lentivirus particles and knockdown of target genes were described previously [19, 20]. The shRNA target sequence for TBX3 was GCA TAC CAG AAT GAT AAG ATA which targeted the coding sequence of both TBX3iso1 and TBX3iso2. The shRNA target sequence for Luciferase (off-target knockdown control) was ACG CTG AGT ACT TCG AAA TGT. The TBX3 shRNA lentivirus particles generated were used to knockdown TBX3 in the 21MT-1 cell line and Luciferase shRNA was used a negative control. One week post-transduction, stable clones were selected in αHE10F containing 0.8 μg/ml puromycin (Sigma-Aldrich) and resistant clones were pooled, expanded, and frozen for later use.
Isolation of RNA and quantitative real-time polymerase chain reaction (qRT-PCR)
Cells were harvested with trypsin and RNA was isolated using the RNeasy Mini Kit (Qiagen). Samples were treated with 30U DNase I (Qiagen), and 500 ng of RNA was converted into cDNA with the Superscript III First-Strand Synthesis System (Invitrogen) using Oligo (dT) 12–18 primers (Invitrogen). qRT-PCR was performed using RT2 SYBR Green ROX qPCR Mastermix (Qiagen,) on an Mx3000P QPCR system. Primers used for total TBX3 were: forward: 5′-CGC TGT GAC TGC ATA CCA GA-3′ and reverse: 5′-GTG TCC CGG AAA CCT TTT GC-3′. Primers used for TBX3iso1 were: forward: 5′-AGT GGA TGT CCA AAG TCG TCA C-3′ and reverse: 5′-CAT GGA GTT CAA TAT AGT AAA TCC ATG TTT GTC TG-3′. Primers used for TBX3iso2 were: forward: 5′-AGT GGA TGT CCA AAG TCG TCA C-3′ and reverse: 5′-CAC TTG GGA AGG CCA AAG TAA ATC CAT G-3′. Primers used for GAPDH were: forward: 5′-AGG CTG GGG CTC ATT TGC AG-3′ and reverse: 5-‘CCA TCC ACA GTC TTC TGG GTG-3′. All primers were purchased from Sigma-Aldrich. Output values were reported relative to GAPDH as fold expression normalized to control cell lines.
Preparation of 21T cell lysates
Radioimmunoprecipitation (RIPA) buffer (10nM Tris-HCl pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 150nM NaCl, 1 % Triton-X 100, 0.5 % DOC, and 1 % SDS) containing one protease inhibitor tablet per 10 ml (Complete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche) was used to lyse cells grown as subconfluent monolayers in 10 cm dishes. The cells were removed with a cell scraper, collected in a clean microcentrifuge tube, and placed on a rotator for 20 min at 4 °C. Tubes were spun at 13,000 RPM for 10 min at 4 °C and the resulting supernatant was collected.
Electrophoresis and Western blotting
Proteins were electrophoresed on 10 % polyacrylamide gels and subsequently analyzed after transfer by Western blot with mouse anti-TBX3 antibody (Abcam, ab58264; 1:2,000), anti-Vimentin (Dako, clone 3B4; 1:1,000), anti-Twist (Abcam, ab50887; 1:1,000), anti-Src (Cell Signaling, 2108; 1:1,000), mouse anti-β-actin antibody (Abcam, ab49900; 1:150,000), and anti-Vinculin (Sigma, V9264; 1:40,000). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-mouse, Amersham GE Healthcare; anti-rabbit, Sigma) diluted 1:10,000, protein bands were detected using ECL Plus Western Blotting Detection Reagents (Amersham GE Healthcare) and then exposed to film in a dark room. Densitometric quantification was performed using ImageJ (Open source software, National Institutes of Health, USA).
Immunohistochemistry of 21T cell pellets
Trypsinized cells were washed twice in phosphate buffered saline (PBS), and pelleted in 15 ml conical tubes. Pellets were re-suspended in 10 % neutral buffered formalin (NBF) and stored at 4 °C overnight. Formalin was removed, and pellets re-suspended in 1 ml of 1 % agarose cooled to 35 °C. The hardened pellets were wrapped in lens paper, cassetted, and processed to paraffin. For histology, 4 μm sections were deparaffinized, pre-treated with 10 mM citrate buffer pH = 6 and blocked for endogenous peroxidises in 3 % H2O2/methanol. Sections were immunostained with rabbit anti-TBX3 antibody (Abcam, ab99302) diluted 1:300 at 4 °C overnight. Signal detection was accomplished using ThermoScientific UltraVision LP Detection System (TL-060-HD).
Three-dimensional Matrigel culture and immunofluorescence
Three-dimensional Matrigel culture was described previously [20–22]. Following a 9 day growth period, images were taken at both 4X and 10X objective using an inverted microscope. ImageJ (Open source software) was used to determine colony formation rates by quantifying the percentage of the population that formed colonies greater than 50 μm in diameter. ImageJ was also used to determine the proportion of circular colonies; a binary quantification method was utilized, with a circular colony having a circularity index above 0.75.
For immunofluorescence of Matrigel cultures, after 9 days of growth, the Matrigel plugs were fixed (10 % NBF) and permeabilized (0.5 % Triton X-100 in PBS). After blocking with normal goat serum (Invitrogen), rabbit anti-Ki67 (Abcam, ab833) and anti-cleaved caspase 3 (Cell Signaling, 9661) antibodies were incubated at 1:150 dilution in 10 % normal goat serum overnight at 4 °C. Cells were incubated with Alexa Fluor 488 Goat Anti-Rabbit secondary antibody (Invitrogen, A11034) and counterstained with Hoechst 33342 (Invitrogen, H1399) and Alexa Fluor 546 Phalloidin (Invitrogen, A22238). Coverslips were mounted over the stained Matrigel plugs with ProLong Gold Antifade Reagent (Invitrogen). Imaging was done using an Olympus Confocal Imaging System (FluoView FV1000 coupled to the IX81 Motorized Inverted System Microscope). Nuclei per colony were manually counted after acquiring 3D colony images.
Transwell migration and invasion assays
To assess the migratory potential of 21NT cells overexpressing TBX3, transwell inserts with 8.0 μm pores (Corning, 3422; 24-well plate) were coated with a thin layer of gelatin (6 μg) to serve as a substrate for migration without obstructing movement through the pores. A 100 μl cell suspension (5 × 105 cells/ml in αHE with 0.1 % bovine serum albumin (BSA)) was added to the upper chambers and 0.75 ml αHE10F media was added to the bottom wells. After incubation at 37 °C and 5 % CO2 for 22 h, migrated cells were fixed with 1 % gluteraldehyde for 20 min and stained with full-strength Hematoxylin for 15 min. Cells that remained on top of the membrane were removed using a cotton swab. Images of 5 non-overlapping fields of view were acquired using Image-Pro Analysis Software (Media Cybernetics) coupled to an inverted microscope at 10X objective. Cells were counted from the acquired images using ImageJ (Open source software). Similarly, to assess the invasive potential, transwell inserts with 8.0 μm pores precoated with Matrigel (Corning, 354480) were used and incubation lasted 22 h. Means derived from four replicates were used during analysis.
RT2 PCR arrays
RNA was isolated from TBX3iso1 and TBX3iso2 transfectants, as well as vector controls, using the RNeasy Mini Kit (Qiagen). One microgram of RNA was converted into cDNA using the RT2 First Strand Kit (Qiagen). RT2 SYBR Green Mastermix was combined with the cDNA and dH2O as per the manufacturer’s protocol. Expression of 84 key genes commonly involved in dysregulation of signal transduction pathways and other biological processes involved in breast cancer were assessed using Human Breast Cancer RT2 PCR arrays (Qiagen, PAHS-131ZA-2). Data analysis was conducted by SA Biosciences PCR Array Data Analysis Web portal using the ΔΔCt method normalized to acidic ribosomal phosphoprotein P0 (RPLP0) expression. Heat map showing absolute expression of mRNA was generated using the SA Biosciences RT2 profiler PCR Array Data Analysis Web Portal.
Statistical analysis
Statistical analyses were done using GraphPad Prism 5.0 software (La Jolla, CA). Colony morphology experiments, stain quantification, migration and invasion assays, and mRNA and protein levels were analyzed using ANOVA followed by Tukey’s post hoc test (for comparison between more than two groups) or Student’s t-test (for comparison between two groups). Proportions were analyzed via Fisher’s exact test. For all analyses, p < 0.05 was considered statistically significant.