LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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CHAPTER 2 MATERIALS AND METHODS the average intensity within the selection (the sum of the intensities of all the pixels in the selection divided by the number of pixels) was measured. All the foci were counted manually; at least 100 cells per experiment were analyzed from three independent experiments. 2.9 Western Blotting Proteins were separated by 8 % SDS-PAGE using minigel apparatus (Technosource, India) run at a constant voltage of 150 volts for 3 hr. The resolved proteins were then transferred to nitrocellulose membrane using an immunoblot apparatus (Technosource, India) at a constant voltage of 70 volts for 90 min. Following transfer, membranes were stained using Ponceau S solution to confirm complete transfer of proteins from the gel. Thereafter, the gel was also stained with 0.2 % coomassie blue to reconfirm the same. The membranes were blocked overnight with wash buffer (0.05 M Tris, 0.15 M NaCl and 0.1 % Tween 20 pH8.0) containing 5 % BSA with gentle rocking at 4 0 C. The blocked membranes were probed with specific primary antibodies (dilutions used are mentioned in the respective figures) followed by washing with wash buffer four times for 5 min each. Thereafter the membranes were reprobed with horse radish peroxidase conjugated secondary antibody (Roche Molecular, Biochemicals. Germany) at a dilution of 1:2000 in wash buffer containing 1 % BSA, washed 4x5 min with wash buffer and developed with Mouse/Rabbit Western Blotting Kit (Roche Molecular, Biochemicals. Germany). Chemiluminescence was recorded on Konica AX medical X-Ray films. Images were digitized using HP Scan Jet ADF Scanner and the figures were assembled using Gelquant Version 2.7 Software. 89
CHAPTER 2 MATERIALS AND METHODS 2.10 Measurement of DNA damage DNA damage in all the groups of EL-4 cells 2 h after exposure to different conditioned medium, was measured by alkaline single cell gel electrophoresis (comet assay) [228]. In brief, frosted microscope slides (Gold Coin, Mumbai, India) were covered with 200 µl of 1% normal melting agarose (NMA) in phosphate buffered saline (PBS) at 45 o C, covered with a cover slip and kept at 4 o C for 10 min. After solidification a second layer of 200 µl of 0.5% low melting agarose (LMA) containing approximately 105 cells at 37 o C was layered over it. The cover slips were replaced immediately and the slides were kept at 4 o C. After solidification of the LMA, the cover-slips were removed and slides were placed in the chilled lysing solution (2.5M NaCl, 100mM Na 2 -EDTA, 10mM Tris–HCl, pH 10, and 1% DMSO, 1% Triton X100 and 1% sodium sarcosinate) for 1 h at 4 o C. The slides were removed from the lysing solution and placed on a horizontal electrophoresis tank filled with freshly prepared alkaline buffer (300mM NaOH, 1mM Na 2 -EDTA and 0.2% DMSO, pH≥13.0) and were equilibrated in the above buffer for 20 min and electrophoresis was carried out at 25V for 20 min. After electrophoresis the slides were washed gently with 0.4M Tris–HCl buffer, pH 7.4, to remove the alkali, stained with 50 µl of propidium iodide (PI, 20 µg/ml) and visualized using a Carl Zeiss Fluorescent microscope (Axioskop). The images (40–50 cells/slide) were captured with high-performance GANZ (model: ZCY11PH 4) color video camera. The integral frame grabber used in this system (Cvfbo1p) is a PC based card and it accepts color composite video output of the camera. The quantification of the DNA strand breaks of the stored images was done using the CASP software by which %DNA in tail, tail length, tail moment and Olive tail moment could be obtained directly [229]. 90
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RADIATION INDUCED SIGNALING IN MAMM
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DECLARATION I, hereby declare that
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CONTENTS Page No. SYNOPSIS………
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2.11 Measurement of NO production
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PREAMBLE SYNOPSIS Ionising radiatio
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SYNOPSIS Aims and Objectives: To St
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SYNOPSIS 2.6 Immunofluorescence sta
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SYNOPSIS ATM gene expression, which
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SYNOPSIS Percentage Survival 100 80
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SYNOPSIS Ratio of Rad52 to Actin Ra
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Relative Phosphorylation 45 40 35 3
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SYNOPSIS Fig. 3.3A Clonogenic Cell
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SYNOPSIS (A) Av No. of phospho BRCA
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SYNOPSIS Percentage Survival 120 10
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SYNOPSIS Fig.3.4B. Existance of cro
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SYNOPSIS Fig. 3.4EDNA damage in EL-
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SYNOPSIS subsequent cytotoxicity ca
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SYNOPSIS [4] Hall, E. J. Radiobiolo
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CHAPTER 1 INTRODUCTION INTRODUCTION
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CHAPTER 1 INTRODUCTION 15.8% 9.56%
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CHAPTER 1 INTRODUCTION far apart an
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CHAPTER 1 INTRODUCTION and are more
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CHAPTER 1 INTRODUCTION 1.3 DNA dama
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Page 76 and 77: CHAPTER 1 INTRODUCTION p90S6 kinase
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Page 84 and 85: CHAPTER 1 INTRODUCTION 1.6 Radiatio
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Page 88 and 89: CHAPTER 2 MATERIALS AND METHODS MAT
Page 90 and 91: CHAPTER 2 MATERIALS AND METHODS 2.2
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Page 96 and 97: CHAPTER 2 MATERIALS AND METHODS PBS
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Page 102 and 103: CHAPTER 3 RESULTS RESULTS 93
Page 104 and 105: CHAPTER 3 RESULTS fractionated regi
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Page 108 and 109: CHAPTER 3 RESULTS Table 3.1.2A List
Page 110 and 111: CHAPTER 3 RESULTS 80 NM_002185 IL7R
Page 112 and 113: CHAPTER 3 RESULTS SAA2 149 AU134977
Page 114 and 115: CHAPTER 3 RESULTS 221 LOC643167 RNA
Page 116 and 117: CHAPTER 3 RESULTS 299 BF244402 FBXO
Page 118 and 119: CHAPTER 3 RESULTS CDK4) 57 AU152107
Page 120 and 121: CHAPTER 3 RESULTS 134 AL045793 METT
Page 122 and 123: CHAPTER 3 RESULTS 58 AF237813 ABAT
Page 124 and 125: CHAPTER 3 RESULTS 127 AI809749 ORMD
Page 126 and 127: CHAPTER 3 RESULTS 31 BE615699 UNC84
Page 128 and 129: CHAPTER 3 RESULTS 47 AF026303 SULT1
Page 130 and 131: CHAPTER 3 RESULTS 117 BF062193 CYor
Page 132 and 133: CHAPTER 3 RESULTS 187 AL036662 ---
Page 134 and 135: CHAPTER 3 RESULTS 52 AV686514 EMP2
Page 136 and 137: CHAPTER 3 RESULTS exposed to 10 Gy
Page 138 and 139: CHAPTER 3 RESULTS Fig.3.1.4 Gene ex
Page 140 and 141: CHAPTER 3 RESULTS Fig. 3.1.6 Radiat
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Page 144 and 145: CHAPTER 3 RESULTS 3.1.9 Stabilizati
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CHAPTER 3 RESULTS whereas only the
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CHAPTER 3 RESULTS Fig.3.2.2 Gene ex
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CHAPTER 3 RESULTS Fig.3.2.4 Gene ex
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CHAPTER 3 RESULTS with equal doses
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CHAPTER 3 RESULTS Fig.3.3.1.2 Forma
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CHAPTER 3 RESULTS Fig.3.3.1.3 Phosp
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CHAPTER 3 RESULTS (7.5±0.64) (Fig.
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CHAPTER 3 RESULTS Fig.3.3.1.7b Phos
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CHAPTER 3 RESULTS 3.3.2 Oxygen beam
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CHAPTER 3 RESULTS in all the cells.
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CHAPTER 3 RESULTS 3.3.2.3 Radiation
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CHAPTER 3 RESULTS Fig.3.3.2.7 Apopt
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CHAPTER 3 RESULTS PMA stimulated U9
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CHAPTER 3 RESULTS Fig.3.4.2. Exista
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CHAPTER 3 RESULTS up-regulation of
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CHAPTER 3 RESULTS Fig.3.4.3.2 NO pr
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CHAPTER 3 RESULTS 3.4.3.4 Apoptotic
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CHAPTER 3 RESULTS 3.4.4 Bystander e
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CHAPTER 3 RESULTS Fig.3.4.4b DNA da
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CHAPTER 3 RESULTS Fig.3.4.5a Gene e
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CHAPTER 4 DISCUSSION DISCUSSION 185
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CHAPTER 4 DISCUSSION directly expos
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CHAPTER 4 DISCUSSION dose is delive
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CHAPTER 4 DISCUSSION A clear cause
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CHAPTER 4 DISCUSSION 4.2 Proton bea
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CHAPTER 4 DISCUSSION Although the e
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CHAPTER 4 DISCUSSION Similar number
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CHAPTER 4 DISCUSSION the mechanism
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CHAPTER 4 DISCUSSION Thus unlike th
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CHAPTER 4 DISCUSSION different from
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CHAPTER 4 DISCUSSION upregulates ma
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CHAPTER 4 DISCUSSION Numerous studi
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CHAPTER 4 DISCUSSION inhibition of
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CHAPTER 4 DISCUSSION The survival o
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CHAPTER 5 REFERENCES [1] Khan, F. T
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CHAPTER 5 REFERENCES [19] Woo, R. A
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CHAPTER 5 REFERENCES [35] Nghiem, P
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CHAPTER 5 REFERENCES [52] McKay, B.
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CHAPTER 5 REFERENCES [69] Cary, R.
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CHAPTER 5 REFERENCES [84] Uziel, T.
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CHAPTER 5 REFERENCES [98] Liu, Y.;
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CHAPTER 5 REFERENCES [111] Fei, P.;
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CHAPTER 5 REFERENCES [127] Frank, K
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CHAPTER 5 REFERENCES [141] Pierce,
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CHAPTER 5 REFERENCES [156] Roots, R
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CHAPTER 5 REFERENCES [171] Xia, Z.;
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CHAPTER 5 REFERENCES (MAP) kinase c
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CHAPTER 5 REFERENCES phosphorylatio
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CHAPTER 5 REFERENCES [211] Anderson
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CHAPTER 5 REFERENCES [229] Konca, K
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CHAPTER 5 REFERENCES [245] Kastan,
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CHAPTER 5 REFERENCES [261] Miralbel
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CHAPTER 5 REFERENCES [278] Weizman,
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CHAPTER 5 REFERENCES [294] Zhou, H.
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PUBLICATIONS AND PRESENTATIONS Publ
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Mutation Research 729 (2012) 61-72
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S. Ghosh, M. Krishna / Mutation Res
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Cancer Invest Downloaded from infor
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1568 I. J. Radiation Oncology d Bio
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