changes in protein profiles in bortezomib applied multiple myeloma ...

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50 50 70 70 50 50 70 70 50 50 70 70 50 50 50 50 50 50 70 70 70 BLANK BLANK 96 Well Plate B Figure 3.1. The Dosage of Bortezomib (nM) Applied on MM U-266 Cells (cont.) In Figure 3.1 ; 70 70 70 C is correspond to control group (100 µl (2x10 4 ) MM U-266 Cells + 100 µl RPMI-1640 Growth Medium), 0.1 is correspond to 0.1 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 0.1 nM Bortezomib), 1 is correspond to 1 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 1 nM Bortezomib), 5 is correspond to 5 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 5 nM Bortezomib), 10 is correspond to 10 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 10 nM Bortezomib), 20 is correspond to 20 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 20 nM Bortezomib), 50 is correspond to 50 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 50 nM Bortezomib), 70 is correspond to 70 nM Bortezomib concentration (100 µl (2x10 4 ) MM U-266 Cells + 100 µl 70 nM Bortezomib), 49
After 72 hour incubation period at 37°C in 5% CO2, they were treated with 50 µl XTT reagent for 4 hours at CO2 incubator. After that, the absorbances of the samples were read under 492 nm wavelength of light by Elisa reader (Thermo Electron Corporation Multiskan Spectrum, Finland) and graphed cell proliferation plots. Finally, IC50 value (the concentration of drug that inhibits 50% of cell proliferation as compared to untreated control) of Bortezomib was calculated from cell proliferation plots. 3.2.6. Evaluation of Apoptosis 3.2.6.1. Measurements of Caspase-3 Enzyme Activity Also called programmed cell death or cell suicide, apoptosis is an integral and necessary part of life cycle of organisms. In the human body, about a hundred thousand cells are produced every second by mitosis and a similar number of them die by apoptosis (Vaux and Korsmeyer, 1999). The apoptotic mode of cell death can be described as a process which plays an important role in the improvement and homeostasis of multicellular organism and in the regulation and maintenance of the cell populations in tissues upon pathological and physiological conditions (Jacobson et al., 1997; Meier et al., 2000; Hengartner, 2000; Leist and Jaattela, 2001). Apoptosis can be triggered by various stimuli from outside or inside the cell. For example, ligation of cell surface receptors, DNA damage as a cause of defects in DNA repair mechanisms, treatment with cytotoxic drugs or irradiation, a lack of survival signals, contradictory cell cycle signaling or developmental death signals (Gewies, 2003). It is doubtless that apoptotic processes are of widespread biological and biochemical significance. A major biochemical feature of apoptosis is the sequential activation of the caspases (cysteine- dependent aspartate- specific proteases), a family of proteases whose substrate include large protein precursors of enzymes capable of destroying DNA (endonucleases); lamin and actin (proteases); and proteins involved in DNA repairing, RNA splicing, signal transduction and transcription factors (Thornberry and Lazebnik, 1998; Hengartner, 2000; Green 2000; King and Robins, 2006). One of the vital members of these caspases family is Caspase-3 enzyme which is the key component in that it takes part in the center of apoptotic pathways (Bratton et 50
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CHANGES IN PROTEIN PROFILES IN BORT
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ACKNOWLEDGEMENTS First of all, with
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ÖZET BORTEZOMĠB UYGULANAN MULTĠP
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2.1.4.1.2. Two-Dimensional Gel Elec
Page 9 and 10: LIST OF FIGURES Figure Page Figure
Page 11 and 12: LIST OF TABLES Table Page Table 1.1
Page 13 and 14: When normal cells in the body begin
Page 15 and 16: Figure 1.2. Development of Cancer S
Page 17 and 18: the myeloma cells does not help fig
Page 19 and 20: A rare form of MM called Nonsecreto
Page 21 and 22: 1.2.3. Risk Factors for MM A risk f
Page 23 and 24: of the mineralizations by adjusting
Page 25 and 26: To measure the levels of red cells,
Page 27 and 28: The levels of blood urea nitrogen (
Page 29 and 30: By using the system introduced by D
Page 31 and 32: 1.2.7.2. Biology of The Proteasome
Page 33 and 34: Figure 1.10. Ubiquitin-Proteasome P
Page 35 and 36: Figure 1.12. Effect of Bortezomib o
Page 37 and 38: encompasses the investigation of pr
Page 39 and 40: of modification. Ubiquitin is a sma
Page 41 and 42: the given cells. On the other hand,
Page 43 and 44: is-acrylamide (N,N'-methylene-bisac
Page 45 and 46: 2.1.4.2. Detection of Protein Spots
Page 47 and 48: ange of 600-2500 Da. This is the id
Page 49 and 50: ions resolved by the mass analyzer
Page 51 and 52: 2.1.4.5.2. Mass Analyzers Figure 2.
Page 53 and 54: There is no doubt that different ma
Page 55 and 56: mask low-quantity proteins that may
Page 57 and 58: 3.1. Materials 3.1.1. Medias CHAPTE
Page 59: 3.2.5. Cell Proliferation Assay Ant
Page 63 and 64: eceptors of the Tumor Necrosis Fact
Page 65 and 66: The preparation of stock BSA Standa
Page 67 and 68: 3.2.6.3. Detection of the Loss of M
Page 69 and 70: centrifugation, the supernatant was
Page 71 and 72: selected symmetrically to load the
Page 73 and 74: approximately 35 ml, the volume of
Page 75 and 76: After electrophoresis was finished,
Page 77 and 78: 7 Day Two: Reduction, Alkylation, a
Page 79 and 80: CHAPTER 4 RESULTS AND DISCUSSION 4.
Page 81 and 82: % Changes in Cytoplasmic/Mitochondr
Page 83 and 84: Table 4.1. Magnified Segments of Di
Page 85 and 86: Table 4.1. Magnified Segments of Di
Page 87 and 88: After labeling, black and red color
Page 89 and 90: 78 7 8 Y2S Y3S Table 4.2. Results o
Page 91 and 92: 80 12 S4Ca Table 4.2. Results of Di
Page 93 and 94: 82 27 Y5S Table 4.2. Results of Dif
Page 95 and 96: Spot 4, Ubiquitin-conjugating enzym
Page 97 and 98: is a transcriptional repressor, but
Page 99 and 100: activity and loss of mitochondrial
Page 101 and 102: distributed, but correlate with nat
Page 103 and 104: DeMartino, G. N. and Slaughter, C.
Page 105 and 106: proteomic analyses of a systematica
Page 107 and 108: Mahindraa, A., Hideshimab, T. and A
Page 109 and 110: Richardson, P. G., Hideshima, T. an
Page 111 and 112:
Terpos, E. 2008. Bortezomib Directl
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A.1. RPMI-1640 Growth Medium APPEND
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Table B.1. Chemicals and Reagents U
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B.4.3. Standard Curve for BSA Table
Page 119 and 120:
Urea (8M) 4.8 g CHAPS (%2) 0.2 g DT
Page 121 and 122:
E. F. G. STOCK REAGENT PREPARATION
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