Material and Methods (Supplementary)
Material and Methods (Supplementary)
Establishment of inducible Notch1 cell line
A 2.3-kb NICD fragment (amino acids 1759 –2556) from pTAN1-cDNA was subcloned into the pRevTRE vector. The cloned construct (pRevTRE-Notch1) was confirmed by DNA sequencing. In brief, the parental FTC236 cells were first transfected with plasmid pRevTet-On (Clontech, Mountain View, CA) containing the Tet-responsive transcriptional activator and selected in medium containing 0.5 mg/mL G418 (Mediatech, Inc, Manassas, VA). The resulting G418-resistant, FTC236-Tet-on clone with the highest inducibility was transfected with either pRevTRE-Notch1or the empty vector (pRevTRE). Transfected cells were selected in 1.0 mg/mL hygromycin (Invitrogen). Resistant FTC236-Notch1 and FTC236-TRE clones were treated with doxycycline and screened for the presence of Notch1 protein by Western blot analysis.
Cell viability assay
FTC236-Notch1 and FTC236-TRE cells were plated in quadruplicate on 24-well plates at a density consistent with exponential growth and incubated overnight. The following day the cells were treated with increasing concentrations of doxycycline up to 1 µg/mL. The cells were then incubated for up to 6 days with subsequent treatments on days 2 and 4. To assess the effect of SERPINE1 knock-down, FTC236 parental cells were plated followed by the transfection with lipofectamine alone, SERPINE1 siRNA or control siRNA the other day. Viable cells were determined by a 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma) after treatment as previously described (19). After the incubation with serum-free medium containing 0.5 mg/mL of MTT at 37°C for 4 hours, DMSO was added to each well and mixed. The results were analyzed at 540 nm using a spectrophotometer (μQuant; Bio-Tek Instruments).
Western Blot
FTC236-Notch1 and FTC236-TRE cells were treated with doxycycline at various concentrations up to 1µg/mL. Samples were collected for protein analysis at different time points of either doxycycline treatment or doxycycline withdrawal. Protein concentration was quantified using the BCA Protein Assay Kit (Thermo Scientific) following the manufacturer’s instructions. Equal amounts of denatured protein were resolved by electrophoresis on 4%–15% Criterion TGX precast gels (Bio-Rad Laboratories), transferred onto nitrocellulose membranes (Bio-Rad Laboratories), blocked in 5% nonfat milk solution, and then immunoblotted with the following primary antibodies overnight at 4°C: Notch1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), SERPINE1 (1:1000, Abcam, Cambridge, MA), cyclin B1(1:1000), cyclin D1 (1:1000), p21Waf1/Cip1(1:3000), β-actin (1:2000, all from Cell Signaling Technology, Danvers, MA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000, Santa Cruz Biotechnology). Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) the following day. The immunoreactive protein bands were visualized by the detection systems of Immunstar (Bio-Rad Laboratories), SuperSignal West Pico or SuperSignal West Femto (Pierce Biotechnology, Rockford, IL). Immunoblot analyses were repeated at least twice and the expression levels of β-actin or GAPDH were used as the loading control.
Clonogenic assay
FTC236-Notch1 and -TRE cells were treated with different concentrations of doxycycline for 48 h prior to plating at a density of 250 viable cells/well. Cells were incubated for 9 days in the absence or presence of doxycycline according to the pre-seeding condition, with media changes every 4 days. In another set of experiment to assess the effect of SERPINE1 knock-down, FTC236 parental cells were plated at a density of 200 viable cells/well 48 hours after the transfection with lipofectamine alone, SERPINE1 siRNA or control siRNA. Cells were allowed to grow for 12 days. Cells were stained with crystal violet (0.05 % w/v in 4 % formaldehyde) (Sigma) and colonies were counted using ImageJ software.
Detection of cell apoptosis by flow cytometry
Cell apoptosis was detected by flow cytometry analysis using PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to manufacturer’s instructions. In brief, FTC236-Notch1 and -TRE cells were seeded in 6-well plate and treated with doxycycline at different concentrations for 48 hours. Cells were harvested and resuspended in binding buffer (10 mM HEPES/NaOH, pH=7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1 × 106 cells/mL. Then PE Annexin V (5 μL) and 7-AAD (10 μL) were added to 100 μL of each samples. Cells were gently mixed and incubated for 15 minutes at room temperature in the dark followed by the addition of another 400 μL of binding buffer. Stained cells were acquired within 1 hour on FACS Calibur (Becton Dickinson) and data were analyzed by FlowJo software (Tree Star).
Cell migration assay
The experiment was carried out using the CytoSelect cell migration assay kit (Cell Biolabs, Inc., San Diego, CA) containing polycarbonate membrane inserts (8 μm pore size) in a 24-well plate. In brief, appropriate amount of cells were suspended in serum-free medium and added to the insert. The lower well was loaded with routine medium (with 10% FBS). After an incubation of 4-6 hours at 37°C in a humidified incubator with 5% CO2, non-migratory cells on the upper surface of the membrane were gently removed by wiping with a cotton swab. The cells that migrated through the pores and to the underside of the membrane were fixed, stained and photographed under a microscope. The number of cells was counted and averaged using three randomly selected areas per membrane. Doxycycline (0.8 µg/mL) was added to medium/cells over the time course of the desired experimental groups.
Wound healing assay
FTC236-Notch1 and FTC236-TRE cells were seeded and grown to confluence on six-well plates. A scratch wound was carefully created in each well using a 200 µl pipette tip. Cells were rinsed with PBS and fresh medium was added with or without doxycycline. Images of the cells were recorded at the time of wound generation and 24 hours after from at least three independent experiments. Photographs were acquired with a microscope using a Zeiss camera and relative wound closure was determined using MRGrab software (Carl Zeiss Microscopy, LLC, Thornwood, NY).
Cell labeling for in vivo study
FTC236-Notch1 and FTC236-TRE cells were transduced with the lentivirus particles expressing firefly luciferase and red fluorescent protein (RFP)(GenTarget Inc., San Diego, CA). Selection was initiated 24 hours post-transduction in both cell lines with 0.5µg/mL puromycin. RFP-positive cells were pooled following fluorescence-activated cell sorting analysis at the University of Wisconsin Comprehensive Cancer Center (UWCCC) Flow Cytometry Core after 7 days of puromycin selection.
Ex vivo imaging and histopathology evaluation
For ex-vivo bioluminescence imaging of dissected lungs, D-luciferin was diluted 1:100 in PBS (final concentration 300 μg/mL) and used to soak freshly dissected lungs for 2 to 3 min before IVIS imaging. Specimens of the xenograft tumors and lungs were fixed in 10% buffered formalin. Samples were then processed and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) according to a standardized protocol. Additionally, immunohistochemistry (IHC) was employed to assess the expression of SERPINE1 with or without Notch induction. Heat-mediated antigen retrieval was performed using citrate buffer pH6.0 prior to staining paraffin sections. After blocking, the sections were incubated with goat polyclonal anti-SERPINE1 antibody (1.7 g/mL, AbD Serotec, Kidlington, UK) at 4°C overnight, and detected using ImmPRESS anti-goat Ig HRP (Vector Labs, Burlingame, CA) for 30 minutes at room temperature. The slides were washed thoroughly with TBS/tween between all stages of the procedures. Finally, the antibody reaction was visualized by color development using the DAB (3,3’-diamino-benzidine tetrachloride) substrate solution (ImmPACT DAB, Vector Labs), and after that, the sections were counterstained with hematoxylin. All stained sections were assessed by a pathologist (R.V.L) for the extension of the primary tumor, the presence of metastases in the lung, and the expression levels of SERPINE1. The intensity of IHC staining was defined as “0” for negative, “1+” for weak, “2+” for moderate, and “3+” for strong staining.
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