Supplemental Figure1.A. RT-PCR showing GATA4 transcript in cDNA isolated from human brain and astrocytes. B. RT-PCR showing GATA4 Gata4 transcript in cDNA isolated from murine brain and murine astrocytes. C. IF negative control for GFAP and GATA4 which are both negative in the human GBM glioma U87 cell line. D. GATA4 siRNA efficiently knocks down GATA4 at the protein level without effecting GATA6 protein levels aftert 48hours of siRNA treatment.
Supplemental Figure 2. MTS cell viability assay as an indirect measure of proliferation. GATA4 knockdown did not confer and cell viability advantages to normal human astrocytes (A) or normal murine astrocytes (B). C.GATA4 Gata4 knockdown in p53-/- null astrocytes increased cell viability compared to untransfected and scramble siRNA controls. *p<.05.
Supplemental Figure 3. TGFB1 stimulates anti-proliferation effect in non transformed astrocytes. A. Human astrocytes stimulated with TGF1 ligand experience reduced proliferation from 24-72 hours by BrdUu assay. B.pP53 -/- null murine astrocytes also experience a reduction in cell proliferation from 24-72 hours post TGF1 stimulation by BrdU assay. C. TGF1 anti-proliferation effect is significantly reduced lost in p53 -/- null murine astrocytes when GATA4 Gata4 is silenced compared to control TGF1 stimulated astrocytes, **pP<.05. Control astrocytes when treated with TGF1 have significant reduction in proliferation compared to non stimulated astrocytes, *pP<.05. D. SMAD3 nuclear translocation 24h post TGFB1 stimulation was used as a positive control to measure effective TGFB1 stimulation. E and F. GATA4 overexpression sensitizes p53 null astrocytes to apoptosis under Ccamptothecin stimulation. Representative PE-Annexin-V plot showing NHAs with empty vector or overexpressing GATA4, both under Ccamptothecin stimulation. M1 peak represents viable cells while M2 represents early + late stage apoptotic cells. To the right, quantification of Annexin-V-PI staining done in triplicate. *p<.0001, **p<.0001.
Acknowledgements:
SA was supported by a RESTRACOMP studentship award from Hospital for Sick Children’s Hospital, and an +??? Others. ??Ontario Institute of Cancer Research award. thanks to other folks not included in the MS???. Thanks to Dr. Deepak Kamnasaran for technical advice and Jing Ma for technical assistance on IHC. AG was funded for this work by the Funds from National Cancer Institute of Canada, Brain Tumor Society and National Brain Tumor Foundation. to AG, funded this work.
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Supplemental Materials and Methods
Immunohistochemistry (IHC) and immunofluorescence (IF)
Paraffin embedded blocks were cut into 5 μm sections and were de-waxed in xylene followed by rehydration in alcohol series. The tissues were subjected to antigen retrieval by pressure cooking for 20 minutes in citrate buffer (pH =6) followed by blocking of endogenous peroxidase in 0.3% H2O2. Specific dilutions of primary antibodies used are as follows: GATA4 monoclonal antibody (Santa Cruz[ceh1] Biotechnology Inc., CA, USA, 1:100), TUJ1 monoclonal antibody (Chemicon International Inc., 1:500), and NeuN antibody (Chemicon International Inc 1:200). Primary antibody incubation times were overnight at 4oC degrees and detection was performed using biotinylated secondary IgG antibodies for 30 minutes using the ABC reagent kit (Vector Labs, CA, USA) and DAB chromagen (Vector Labs). Sections were counter-stained briefly in hematoxylin (Fisher Scientific Inc., Canada) for 30 secs, dehydrated in 70, 80 and 100% ethanol series, followed by a brief wash in xylene and mounted in Permount (Fisher Scientific Inc.). Hemotoxylin and Eosin (H&E) sections were stained using standard protocols (Eosin Yellowish Solution 1% w/v, Fisher Scientific Inc.)
For double immunofluorescence, 5 x 103 murine and human astrocytes were grown on coverslips in 6-well media plates supplement with D-MEM(Wisent) and 10%FBS (Wisent). The following day, cells were fixed in 4% PFA (Paraformaldhyde, Pierce Chemical Co., Rockford, IL) [ceh2] and incubated with GATA4 monoclonal antibody (Santa Cruz Inc., 1:50), and GFAP rabbit polyclonal (DAKO International Inc., 1:500). Detection was carried out by using fluorescent labeled secondary antibodies (Alexa Fluor 488 and Alexa Fluor 588, Invitrogen Inc). Cells were also stained with DAPI (Vector with DAPI mounting medium). Images were captured with Nikon ACT-1 software and a Nikon E-600 microscope mounted with a digital camera and fluorescence lamp.
Real time Primer Sequences
Primer Name / Sequence 5’3’Gata4 Forward / GCT CCT TCA GGC AGT GAG AG
Gata4 Reverse / CTG TGC CCG TAG TGA GAT GA
Bax Forward / GCT GTT GGG CTG GAT CCA AG
Bax Reverse / TCA GCC CAT CTT CTT CCA GA
Apaf1 forward / AAC CAG GAT GGG TCA CCA TA
Apaf1 reverse / ACT GAA ACC CAA TGC ACT CC
Bcl-2 forward / GCA ATT CCG CAT TTA AAT CAT GG
Bcl-2 reverse / GAA ACA GGC CAC GTA AAG CAA C
B-actin forward / TTG TTA CAG GAA GTC CCT TGC C
B-actin reverse / ATG CTA TCA CCT CCC CTG TGT G
Hprt1 forward / TGA CAC TGG CAA AAC AAT GCA
Hprt1 reverse / GGT CCT TTT CAC CAG CAA GCT
Real time PCR Analysis
Real-time PCR data was analyzed using Opticon Monitor 3.1.3 analysis software. For each sample, a normalization factor (NF) was calculated with the geometric mean of the relative quantities of all reference genes (-actin and HPRT1). The expression level for each sample was then obtained using the equation: expression level = relative quantity/ NF.
Western Blot Analysis
Astrocytes cultured in vitro were lysed with standard PLC lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich Inc.). Protein concentration was determined using the BCA(bicinchoninic acid) [ceh3]assay (Pierce Chemical Co., Rockford, IL). 30ug of protein lysate were loaded into 10% or 12% SDS-PAGE gels. Proteins were then transferred onto PVDF membrane (NEN Research Products) using a semi-dry transfer apparatus (Bio-Rad). Membranes were probed for varying proteins at one hour: GATA4 (G4; Santa Cruz Inc., 1:200), -actin (Sigma-Aldrich Inc., 1:5000), p15INK4B (K-18, Santa Cruz Inc., 1:200), Cyclin D1 (Santa Cruz Inc., 1:400), Cyclin D2(Abcam, 1:400), p16INK4A (p16) (Santa Cruz Inc.), p14ARF (Santa Cruz Inc., 1:200), p19Arf (Santa Cruz Inc., 1:200) and p53 (Vectorshield laboratories, 1:300). Following incubation, membranes were washed and incubated with horseradish peroxidase-conjugated antibodies against the species the primary antibody was raised against (BioRad Laboratories, Inc., CA, USA). Protein detection was carried out by using Chemiluminescence Reagent Plus (PerkinElmer Inc., Massachusetts, USA).
Flow Cytometry
Briefly, 5 x 105 murine astrocytes and 1 x 106 human astrocytes were treated with siRNA or plasmid to knockdown or overexpress GATA4. Untransfected[ceh4] and siRNA negative control treatments were used as negative controls. After 48 hrs of transient transfection, the cells were harvested and re-suspended in 50l of PBS/Hanks Buffered Saline solution plus 2% fetal calf serum, followed by fixation in 1ml of 80% ice-cold ethanol for 30mins. The cells were treated with 50g of Propidium Iodide in 0.6% NP-40 plus 100mg of RNAseA for 30mins at room temperature, prior to filtering through 85m Nitex meshes. The cells were subjected to Flow Cytometry with a FACScan machine (BD Biosciences) and the data was analyzed with the Cell Quest Pro software (BD Biosciences).
In vitro proliferation assay
Proliferation was measured using the Cell Proliferation ELISA, BrdU (chemiluminescent) assay© (Roche Pharmaceuticals). Briefly, the assay is based on the detection of BrdU incorporated into the genomic DNA of proliferating cells. 5x103 cells were seeded and grown in 96-well tissue-culture microplates and subjected to either siRNA or GATA4 plasmid treatment. At appropriate time points, cells were administered BrdU for 12 hours at a final concentration of 10um. After removing the labeling medium, cells were fixed by adding FixDenat (fixing buffer). After removal of FixDenat, anti-BrdU-POD antibody was added and the immune complexes and reactions were quantified using a multi-well luminometer.
Apoptosis measurement
Apoptosis was induced by administration of 10uM of Camptothecin (Sigma-Aldrich) or by 5Gray(Gy) irradiation. 5 x 103 cells were seeded into a 96-well plate, transfected with siRNA, and at 48 hours post-transfection were administered Camptothecin or 5Gy irradiation. Caspase 3/7 activity levels were measured using the Apo-One® Homogeneous Caspase 3/7 assay (Promega Corp., USA) that provides a profluorescent substrate and a cell lysis/activity buffer for Caspase 3/7 (DEVDase) activity. After induction of apoptosis, 100uL of Apo-One was seeded in each well, incubated for 3 hours and then fluorescence levels measured (485Ex/527Em).
PE Annexin V Apoptosis Detection (BD Pharminigen kit (559763))
Briefly, 106 cells in triplicate were plated, NHAs, NHAs + GATA4, p53 null astrocytes, p53 + Gata4. 10 uM Camptothecin was added to induce apoptosis and cells harvested 12h post camptothecin treatment. Cells were rinsed twice with cold PBS and then resuspended 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml. 100 μl of the solution (1 x 10^5 cells) was transferred to a 5 ml culture tube. 5 μl of PE Annexin V and 5 μl 7-AAD were added. Cells were incubated for 15 min at RT (25oC)in the dark. 400 μl of 1X Binding Buffer was added to each tube and analyze by flow cytometry within 1 hr.
ChIP assay
ChIP assays were performed as described previously(Shang et al., 2002). GATA4 antibody (G4; Santa Cruz Biotechnology Inc.) and normal murine IgG (Santa Cruz Inc.) were used for immunoprecipitation (2ug Antibody for 500ug of protein lystate). PCR for human INK4Bp15 promoter was performed with the following primers: forward, 5′-AGC ACC ACA CCC CTC TCT TA-3′; reverse, 5′-TCT GAG ATC CCA GTT TCA TCA-3′. PCR primers for the murine p15Ink4b promoter were as follows: forward, 5′-TTT AAT GAC AGG CCC AGC AC-3′; reverse, 5′-GAC GTG TGC CTG TAC TCA CC-3′. For the distal negative control GATA site at -2949bp for the human, the following primers were used: forward 5’-TTG CTG GCA AAT ATC AGA CG-3’ and reverse 5’-GCA AGA CCA TCA CCC TTG AG-3’. For the -2207bp distal negative control GATA site in the mouse promoter, the following primer pairs were used: forward, 5’-GAG AGA GAG AGA GAG AGA GAG AGA GA-3’ and reverse 5’-TGA TGA TAG ATT TTA GTG TGC TGT TTT-3’. Promoter sequences were obtained from UCSC genome browser ( and Transcriptional Regulatory Element Database (TRED) (Cold Spring Harbor,
1
[ceh1]You need to give the city and country source for all reagents.
[ceh2]Define all abbreviations
[ceh3]define
[ceh4]shouldn’t you use scrambled siRNA as a control?