• Molecules and Cells
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• 제25권2호
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• pp.184-195
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• 2008

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP ($c-FLIP_L$), but not the short form ($c-FLIP_S$), enhanced adhesion and motility. Downregulation of $c-FLIP_L$ with siRNA decreased phosphorylation of FAK and ERK, while overexpression of $c-FLIP_L$ increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed $c-FLIP_L$-promoted motility. Inhibition of ROCK by Y27632 suppressed the $c-FLIP_L$-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of $c-FLIP_L$ increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced $c-FLIP_L$- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the $c-FLIP_L$-promoted cell motility. We conclude that $c-FLIP_L$ promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2251-2256
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• 2015

To study effects of cellular FLICE (FADD-like IL-$1{\beta}$-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinduced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

• Bulletin of the Korean Chemical Society
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• 제27권1호
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• pp.87-92
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• 2006

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8. It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. However, the interaction of FADD and $c-FLIP_s$ (short form) in apoptosis signaling has been controversially discussed. We show the purification and the characterization of human full-length FADD and $c-FLIP_s$ expressed in Escherichia coli. The purified FADD and $c-FLIP_s$ are shown as homogeneity, respectively, in SDS-PAGE analysis and light-scattering measurements. The folding properties of the $\alpha$-helical structure of FADD and the super-secondary structure of $c-FLIP_s$ proteins were characterized by circular dichroism spectroscopy. Furthermore, we report here a series of biochemical and biophysical data for FADD-$c-FLIP_s$ binding in vitro. The binding of both FADD and $c-FLIP_s$ proteins was detected by BIAcore biosensor, fluorescence measurement, and size-exclusion column (SEC).

• BMB Reports
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• 제47권9호
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• pp.488-493
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• 2014

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.

• Asian-Australasian Journal of Animal Sciences
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• 제23권3호
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• 마이크로전자및패키징학회지
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• 제15권4호
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• pp.9-15
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• 2008

Au와 Sn을 순차적으로 도금한 Au/Sn 범프를 플립칩 본딩하여 Au-Sn 솔더 접속부를 형성 후, 미세구조와 접속저항을 분석하였다. $285^{\circ}C$에서 30초간 플립칩 본딩한 Au-Sn 솔더 접속부는 $Au_5Sn$+AuSn lamellar 구조로 이루어져 있으며, 이 시편을 $310^{\circ}C$에서 3분간 유지하여 2차 리플로우시 $Au_5Sn$+AuSn interlamellar spacing이 증가하였다. $285^{\circ}C$에서 30초간 플립칩 본딩한 Au-Sn 접속부는 15.6 $m{\Omega}$/bump의 평균 접속저항을 나타내었으며, 이 시편을 다시 $310^{\circ}C$에서 3분간 유지하여 2차 리플로우 한 Au-Sn 접속부는 15.0 $m{\Omega}$/bump의 평균 접속저항을 나타내었다.

• 한국마이크로전자및패키징학회:학술대회논문집
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• 한국마이크로전자및패키징학회 2003년도 International Symposium
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• pp.253-257
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• 2003

The low temperature flip chip technique is applied to the package of the temperature-sensitive devices for LCD systems and image sensors since the high temperature process degrades the polymer materials in their devices. We will introduce the various low temperature flip chip bonding techniques; a conventional flip chip technique using eutectic Bi-Sn (mp: $138^{\circ}C$) or eutectic In-Ag (mp: $141^{\circ}C$) solders, a direct bump-to-bump bonding technique using solder bumps, and a low temperature bonding technique using low temperature solder pads.

• 마이크로전자및패키징학회지
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• 제17권3호
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• pp.27-32
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• 2010

Cu pillar 범프와 Sn 패드로 구성된 플립칩 접속부를 형성한 후, Sn 패드의 높이에 따른 Cu pillar 플립칩 접속부의 열 싸이클링 및 고온유지 신뢰성을 분석하였다. Cu pillar 플립칩 접속부를 구성하는 Sn 패드의 높이가 5 ${\mu}m$에서 30 ${\mu}m$로 증가함에 따라 접속저항이 31.7 $m{\Omega}$에서 13.8 $m{\Omega}$로 감소하였다. $-45^{\circ}C{\sim}125^{\circ}C$ 범위의 열 싸이클을 1000회 인가한 후에도 Cu pillar 플립칩 접속부의 접속저항의 증가가 12% 이하로 유지되었으며, 열 싸이클링 시험전과 거의 유사한 파괴 전단력을 나타내었다. $125^{\circ}C$에서 1000 시간 유지시에도 Cu pillar 플립칩 접속부의 접속저항의 증가가 20% 이하로 유지되었다.

• Nuclear Engineering and Technology
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• 제11권1호
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• pp.29-45
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• 1979

이 논문에서는 6개의 다른 연료봉 모형을 기준으로 하여 TRIGA Mark-III 연구용 원자로의 핵특성이 자세히 분석되고 있다. 현재 핵연료로 사용하고 있는 FLIP fuel은 연료의 수명을 증진시키기 위해 개발되었던 모형이나, 70%의 고농축이기 때문에 비확산금지조약에 위배되어 수입이 곤란한 실정이다. 따라서 본 논문에서는 FLIP 연료 모형에 대치할 수 있는 다른 연료봉온 찾고 그들의 핵특성을 비교함으로써 앞으로 연료수입계륵을 설립하고자 한다.

• 전자공학회논문지C
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• 제35C권1호
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• pp.10-14
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• 1998

In this paper a double edge triggered (DET) filp-flop is proposed which changes its output state at both the positive and the negative edge transitions of the triggering input. DET filp-flop has advantages in terms of speed and power dissipation over single edge triggered (SET) filp-flop has proposed DET flip-flop needs only 12 MOS transistors and can operate at clock speed of 500 MHz. Also, the power dissipation has decreased about 33% in comparison to SET flip-flop.