LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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Figure 4.3.17: 14-3-3γ loss loss does not lead to alteration in the localization of keratin 8 and keratin 18. HCT116 derived vector control or 14-3-3γ knockdown clones were fixed and immuno stained with the indicated antibodies (Keratin 8 and Keratin 18). Please note that localization of both Keratin 8 and Keratin 18 are not altered in 14-3-3γ knockdown clones as compared to vector control cells. . (Original magnification x 630 with 2X optical zoom). 4.3.8 Reintroduction of shRNA resistant 14-3-3γ in 14-3-3γ knockdown clones rescues the defects in the localization of desmosomal proteins. To ensure that the defects in the localization of the desmosomal proteins observed were not due to off target effects of the shRNA constructs, rescue experiments were performed with a shRNA resistant version of 14-3-3γ, 14-3-3γ-R. HCT116 derived 14-3-3γ knockdown clones were transfected with either GFP or GFP 14-3-3γ-R mutant constructs. These cells were then stained using antibodies to PG. As shown in fig 4.3.18, expression of GFP 14-3-3γ-R in the HCT116 derived 14-3-3γ knockdown clones resulted in a re-localization of PG to cell border, a result that was not observed in cells transfected with GFP alone (Figure 4.3.18). These results suggest that the defects in the localization of desmosomal proteins are specifically due to the loss of 14-3-3γ. 136
Figure 4.3.18: Reintroduction of shRNA resistant 14-3-3γ in 14-3-3γ knockdown clones rescues the defects in the localization of desmosomal proteins. (A) HCT116 derived 14-3-3γ knockdown clones were transfected with either constructs expressing GFP or GFP-14-3-3γ-R mutant, cells were fixed 60 hrs post transfection and immuno stained with antibodies to PG. White arrows show the transfected cells and the yellow arrows show untransfected cells. Please note that GFP-14-3-3γ-R is able to rescue the localization of PG in 14-3-3γ knockdown clones. In contrast, GFP alone is not able to rescue the localization of PG in 14-3-3γ knockdown clones. . (Original magnification x 630 with 2X optical zoom). 137
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Generation of knockdown mice that l
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STATEMENT BY AUTHOR This dissertati
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CERTIFICATE I certify that the thes
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I am thankful to Mr Uday Dandekar f
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3.16 Isolation of Genomic DNA (gDNA
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A synopsis of thesis to be submitte
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esults in loss of 14-3-3 binding an
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and NheI (NEB) to remove tetO-CMV,
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Hanging drop and wound healing assa
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360). To generate transgenic animal
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asement of the seminiferous tubules
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normal female, pups were screened f
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14-3-3ε only in the presence of HP
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proteins are expressed in testis (3
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5. Lalit Sehgal, Srikanth B., Khyat
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Presented a poster at 33rd All Indi
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shRNA siRNA TBS-T U.V WT C IF DSG D
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List of Figures: Page No 1. Figure
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37. Figure 4.3.20: 14-3-3γ interac
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CHAPTER 1 INTRODUCTION 39
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1.1.1 Cyclin Cdk complexes Eukaryot
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1.1.1.c Cyclin A cdk complexes The
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of which are associated with acquis
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1.2.2 The G1/S DNA Damage checkpoin
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complexes. DNA damage induced degra
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cytoplasmic accumulation of cdc25 p
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14-3-3 binding site. 14-3-3 binding
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G2 arrest and induced premature chr
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emoval of S216 phosphorylation as i
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embryogenesis, a loss of cdc25A can
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dimer was essential for Raf activat
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of enhanced apoptosis of peripheral
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the generation of transgenic mice a
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CHAPTER 2 AIMS AND OBJECTIVES 67
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CHAPTER 3 MATERIALS AND METHODS 69
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Name of oligonucleotide EF1α Forwa
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cleavage sites GAAGGTATATTGCTGTTGAC
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R E1 A GAACGAATAGTGAAGCCACAGATGTA s
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100mm 25ug 225ul 250ul 500 1000ul T
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for the transgene were considered a
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solution in TBS-T containing 0.02%
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mounting agent. Confocal images wer
Page 85 and 86: 3.26 Antibodies used for Western bl
Page 87 and 88: each plate was added cold 500ml of
Page 89 and 90: 3.32 Hanging drop assay to determin
Page 91 and 92: 3.37 Biodistribution of 18 F -FDG u
Page 93 and 94: 2.5M NaCl 150mM 60ml Tween-20 (Sigm
Page 95 and 96: CHAPTER 4 RESULTS 95
Page 97 and 98: Figure 4.1.1 Generation of lentivir
Page 99 and 100: 4.1.2 Generation of lentiviral vect
Page 101 and 102: transduction with the virus. Untran
Page 103 and 104: Figure 4.1.6: Application of bi-cis
Page 105 and 106: Figure 4.1.7: Application of bi-cis
Page 107 and 108: Figure 4.2.1: Infection of morulae
Page 109 and 110: Figure 4.2.2: Generation of transge
Page 111 and 112: Figure 4.2.3: EGFP-f expression in
Page 113 and 114: sequenced and the distribution amon
Page 115 and 116: 4.3 Generation of 14-3-3γ knockdow
Page 117 and 118: numbering). Alignment was done usin
Page 119 and 120: presence of EGFP-f by performing po
Page 121 and 122: the mouse tested infected with vect
Page 123 and 124: Figure 4.3.7: Staging of 14-3-3γ k
Page 125 and 126: containing spermatozoa is on the Y-
Page 127 and 128: Figure 4.3.9: 14-3-3γ knockdown le
Page 129 and 130: Figure 4.3.11: 14-3-3γ knockdown i
Page 131 and 132: intercellular space (12, 110, 115,
Page 133 and 134: Figure 4.3.13: 14-3-3γ loss leads
Page 135: Figure 4.3.16: Localization of PKP3
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Page 141 and 142: from our laboratory demonstrated th
Page 143 and 144: Figure 4.3.21: Plakoglobin recruitm
Page 145 and 146: 4.3.23 (A and B)). These proteins g
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Page 149 and 150: Figure 4.3.25: Generation of stable
Page 151 and 152: KIF5B, however its reduction does n
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Page 155 and 156: 4.4 Generation of 14-3-3ε knockdow
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Page 159 and 160: Figure 4.4.3: 14-3-3ε knockdown in
Page 161 and 162: Figure 4.4.4: Patchy hair loss in 1
Page 163 and 164: follicle formation marked by black
Page 165 and 166: Figure 4.4.7: Phenotypes in 14-3-3
Page 167 and 168: compared to WT lungs (indicated by
Page 169 and 170: compared to wild type (Figure 4.4.1
Page 171 and 172: and CD4 positive lyumphocyte number
Page 173 and 174: Figure 4.4.13: Bone marrow cells fr
Page 175 and 176: Figure 4.4.15: Generation of stable
Page 177 and 178: Figure 4.4.16: NIH3T3 cells lacking
Page 179 and 180: Figure 4.4.18: Generation of induci
Page 181 and 182: CHAPTER 5 DISCUSSION 181
Page 183 and 184: to generate transgenic animals expr
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(reviewed in (152, 374)). Kinesins
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subtypes known to date. Type IV col
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Figure 5.1 Schematic representation
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loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
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364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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