Plasmids, transient transfection, and site directed mutagenesis
Cells were centrifuged at 194 g for 5 minutes and genomic DNA was isolated using Qiagen
DNA isolation kit (Qiagen, Valencia, CA). To generate various 5’ deletion constructs of CD133
promoter upstream of transcription start site, sequences were amplified from genomic DNA
isolated from NSC23 cells using appropriate primers. Luciferase constructs driven by the CD133
promoter were generated using a two step protocol. The genomic DNA fragment corresponding
to the CD133 promoter region was PCR amplified using primers incorporating a Kpn1 (forward
primer) and HindIII (reverse primer) restriction sites at the 5’ ends and subsequently cloned into
pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA). The insert from the positive colonies was
obtained by simultaneous restriction digestion using Kpn1 and HindIII restriction enzymes (New
England Biolabs, Ipswich, MA) and subcloned into pGL4.16 [luc2CP/Hygro] vector (Promega,
Madison, WI) which had been linearized by digested with same restriction enzymes followed by
treatment with calf intestinal alkaline phosphatase (CIP) (New England Biolabs, Ipswich, MA).
The primers used to prepare the promoter constructs are listed in Table 1. For transient
transfection experiments, cells were seeded at 5x104 cells/well in 12 well plates 24 h prior to
transfection. The promoter constructs (0.5 g) and beta actin promoter driven Renilla luciferase
were co-transfected in triplicate using Fugene HD (Roche, Indianapolis IN) according to the
manufacturer’s instructions with a 3:1 ratio of transfection reagent to DNA. Cells were lysed
with 100l passive lysis buffer (Promega, Madison, WI) 24hrs after transfection and the cells
lysates were centrifuged to sediment cell debris. Luciferase assay was measured in 20l aliquots
of the lysates using the Dual Luciferase Reporter (DLR) assay system (Promega, Madison, WI)
according to the manufacturer’s instructions. Plasmids for all the transfection experiments were
purified using Qiagen plasmid midi prep system.
Quantitative PCR and Semi-quantitative PCR
RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was
performed using total RNA for each cell line which was reverse transcribed using enhanced
Avian HS RT-PCR Kit ( Cat# HSRT20 or HSRT 100 ) from Sigma. Briefly, 2ug total RNA with
2uM anchored oligo (dT) 23 in 10ul volume for each cell line was heated to 70°C for 10 minutes
followed by 2 minutes incubation on ice in a thin walled 200 l tube. A 10 l mixture including
2 l 10x AMV-RT buffer, 2 l 10mM dNTP mix, 1l RNase inhibitor, 1 l Enhanced avian RT
and 4 l ddH2O was added to denatured RNA . The reaction tubes were incubated at 50°C for 50
minutes, 85°C for 5 minutes then the cDNA was processed for PCR. CD133 cDNA or GAPDH
cDNA was amplifies using the JumpStart REDTaq® Ready Mix from Sigma (cat# P0982). The
sequence of RT PCR primers used to amplify CD133 transcript are as follows - forward primer
5’-GCCACCGCTCTAGATACTGC-3’ and reverse primer 5’-TGTTGTGATGGGCTTGTCAT3’. For each 25 l reaction volume, 12.5 l of 2x master mix, 1 l of 10 M primer, 2 l of
cDNA aliquot, and 9.5 l ddH2O were mixed in a thin walled 200ul tube. The reactions were
conducted in a BioRad thermal cycler with initial denaturation at 95°C for 2 minutes, followed
by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds and then a final
extension at 72°C for seven minutes.
Electrophoretic Mobility Shift Assay (EMSA)
EMSA was performed using a non radioactive based assay (Thermo Scientific Life Science,
Rockford IL). The 5’ biotin labeled oligonucleotides (20 fmol) containing the putative Sp1
binding sites were incubated in the presence of nuclear extracts prepared from NSC23 cells,
binding buffer (10mM Tris pH7.5, 50mM KCL, 1mM DTT), glycerol (2.5%), MgCl2 (5mM),
poly-dI.dC (50ng/ul), NP40 (0.05%) at room temperature for 30 minutes. The binding reaction
was analyzed by PAGE (4% acrylamide gel, 0.5X Tris/Borate/EDTA buffer pH 8.8). Following
electrophoresis, the protein DNA complex was transferred onto a nylon membrane (Bio-Rad)
using a tank transfer apparatus in 0.5X TBE buffer pre cooled to 4oC at 380 mA for 30 minutes.
After UV fixation using a UV cross linker (Stratagene), the membrane was probed using
horseradish peroxide conjugated streptavidin and the reaction analyzed per manufacturer’s
instructions. The sequences of the oligonucleotides used for the assay are: forward 5’ BiotinATTCGATCGGGGCGGGGCGAGC- 3’, reverse 5’ BiotinGCTCGCCCCGCCCCGATCGAAT -3’. Unlabelled oligonucleotides containing consensus Sp1
binding site (Promega) were used for competition studies.
Chromatin Immunoprecipitation (ChIP) Assay
ChIP assay was performed using EZ-ChIP chromatin immunoprecipitation kit (Millipore,
Billerica, MA). Briefly, NSC23 and U251HF cells used for the assay were incubated in 1%
formaldehyde for crosslinking for 10 min. The reaction was stopped by incubating in 125mM
glycine for 5 min. After a wash in PBS, the cells were collected in 2 ml ice cold PBS
supplemented with Protease Inhibitor cocktail (kit) by gently scraping and lysed in 1 ml SDS
lysis buffer containing a protease inhibitor cocktail. The lysates were sonicated to shear the DNA
to a size of approximately 100bp to 500bp. After centrifugation, 20 l of the lysate was diluted
using 180 l dilution buffer in a 0.5 ml microfuge tube. Following dilution the lysate was precleared using 15 l Protein G agarose (Kit) for 1 hour at 4oC. The precleared lysate was
incubated with 2 g of antibody and corresponding IgG control overnight at 4oC. Following day
the immunoprecipitates were recovered by incubating the solution in 20 l protein G agarose for
1 hour at 4oC. After centrifugation, the beads were successively washed in salt buffers. The DNA
was recovered by two successive elutions using elution buffer (0.1M NaHCO3, 1%SDS). The
eluted complexes were incubated at 65oC in NaCl (200 mM) to reverse the crosslink, and the
DNA was extracted by RNase A treatment and proteinase K digestion. The DNA was
subsequently purified using spin columns and eluted in a final volume of 25 l.CD133 promoter
DNA fragments were detected by PCR using the primers shown (Table 3). The DNA samples
were heated to 98oC for 30 seconds followed by 30 cycles of heating to 98oC for 10 seconds,
annealing at 58oC for 20 seconds and extension at 72oC for 10 seconds.