우리 지 시 Borok와 고헤이 Miyazono 원고 준비에 대 한 감사. 당사와 수소 우에하라 기념 재단 연구 친교에 의해 지원 되 고 당사 해외 유학에 대 한 수정 Hayaishi 기념 장학금에 의해 지원 됩니다. 이 작품은 다케다 과학 재단 (A.S.)에서 교부 금에 의해 지원 되었다.

1입니다. EndMT의 유도
<ol><li>표준 문화 조건에 MS-1 세포를 유지 하 고 confluency을 피하기 위해. MS-1 셀의 소스 테이블의 자료에 설명 되어 있습니다. MS-1 셀에 10% 태아 종 아리 혈 청 (FCS), 50 U/mL 페니실린, 및 50 μ g/mL 스 최소 필수 매체-α (MEM-α)를 사용 합니다.</li>
	<li>10 cm에 씻어 MS-1 셀 버퍼링 하는 인산 염 (PBS) x 1 접시 하 고 접시에 trypsin의 1.0 mL를 추가 합니다. 37 ° c.에 5 분 동안 품 어</li>
	<li>문화 미디어의 9 mL를 사용 하 여 셀을 분리 합니다. 15 mL 튜브에 세포 현 탁 액을 수집 합니다.</li>
	<li>실 온에서 5 분 동안 300-400 x g 에 세포 현 탁 액 원심</li>
	<li>조심 스럽게는 상쾌한을 제거 하 고 미리 데워 진된 문화 미디어에 셀 펠 릿을 중단.</li>
	<li>Trypan 블루 솔루션 및 표준 hemocytometer 또는 자동 셀 카운터를 사용 하 여 가능한 셀을 계산 합니다.</li>
	<li>긴 기간 문화에 대 한 c m2 당 103 셀 x 0.5에서 코팅된 표준 문화 접시에 접시 MS-1 셀. 예를 들어 당 6에서 잘 플레이트 5.0 x 103 셀 잘 격판덮개 문화. FCS의 동일한 농도 포함 하 여 동일한 문화 매체를 사용 합니다. MS-1 셀, TGF-β FCS의 사용으로 장기 문화 (적어도 48-72 h)에 EndMT을 유도합니다.</li>
	<li>24 시간 후 TGF-β2 (1 ng/mL의 최종 농도)와 내 피 세포를 자극.</li>
	<li>(5% CO2, 37 ° C) 습도 인큐베이터에 문화 세포. 때 형태학 변화에 초점을 맞추고 세포를 매일 모니터링 합니다.</li>
	<li>장기적인 문화에서 TGF-β와 치료의 48 h 후 TGF-β2를 포함 하는 신선한 미리 데워 진된 문화 미디어에 미디어를 바꿉니다.</li>
	<li>다운스트림 분석을 진행 합니다. 일반적으로, 말라 개편 후 치료, 24 h 관찰 하 고 내 피 마커의 downregulation 및 엽 마커의 upregulation TGF-β와 치료의 48-72 시간 후 관찰 된다.</li>
</ol>2. Immunocytochemical 분석
<ol><li>MS-1 셀 표준 문화 조건에 유지 합니다.</li>
	<li>플레이트 MS-1 셀 4에 잘 잘 (1.7 cm2) 당 103 셀 x 1.0에서 문화 챔버 슬라이드 세포. 슬라이드는 미리 젤라틴 코팅. 잘 당 문화 미디어의 사용 0.5-1.0 mL.</li>
	<li>24 시간 후 TGF-β2 (1 ng/mL의 최종 농도)와 내 피 세포를 자극. 분석할 때 EndMT에 바위의 참여, TGF-β 치료 록 억제제 Y-27632 (10 μ M) 1 시간 전에 추가 합니다. TGF-β 신호 억제의 효과 평가할 때는 TGF-β 수용 체 키 니 아 제 억제제를 사용할 수 있습니다.</li>
	<li>(5% CO2, 37 ° C) 습도 인큐베이터에 문화 세포. 말라 개편 TGF-β와 치료의 24 h 후 MS-1 셀에서 관찰 됩니다. 다른 변경 후 48-72 h 치료의 명백한 될. 72 h 문화에 대 한 신선한 미디어 TGF-β와 치료의 48 h 후 TGF-β를 포함 미디어를 교체 합니다.</li>
	<li>
F-말라 얼룩
	<ol>
<li>TGF-β 치료의 24 h 후 미디어를 제거 하 고 실 온에서 20 분에 대 한 1 x PBS에 4 %paraformaldehyde 셀 수정 1 x PBS 가진 세포를 씻어.</li>
		<li>실 온에서 5 분에 대 한 Triton X-100 0.2% 품 어.</li>
		<li>3 번 1 x PBS와 셀 린스.</li>
		<li>Phalloidin tetramethylrhodamine B isothiocyanate 버섯 phalloides 희석 150-fold에서 함께 품 어 실 온에서 1 h에 대 한 차단 버퍼.</li>
		<li>3 번 1 x PBS와 셀 린스.</li>
		<li>실 온에서 5 분 동안 cyanine 핵 산 바인딩 염료와 핵 얼룩.</li>
		<li>린스 슬라이드 마운트 coverslips 미디어를 탑재 하는 슬라이드를 사용 하 여 슬라이드에 얼굴.</li>
		<li>Confocal 현미경으로 관찰 합니다. 543 nm 레이저와 560-615 nm의 방출 필터를 사용 하 여 phalloidin의 형광의 탐지를 위해. 633 nm 레이저와 650 이상 방출 필터를 사용 하 여 핵 얼룩에 대 한 nm. TGF-β와 치료, 시 두꺼운 스트레스 섬유의 형성 관찰 될 것 이다.</li>
	</ol>
</li>
	<li>
VE cadherin와 α-SMA 얼룩
	<ol>
<li>TGF-β 치료의 72 h 후 미디어를 제거, 감기 50% 메탄올 및 50% 아세톤 셀 수정 (0.5-1.0 mL 잘 당) 5 분.</li>
		<li>3 번 1 x PBS와 셀 린스 (0.5-1.0 mL 잘 당).</li>
		<li>제조업체의 권장된 농도 따라 어둠 속에서 4 ° C에서 하룻밤 블로킹 버퍼에 1 차 항 체로 품 어. 사용 하 여 VE cadherin 단일 클론 항 체 (BV13, 1: 100 희석)와 α-SMA Cy3 활용 된 단일 클론 항 체 (1A4, 1: 200 희석)20.</li>
		<li>3 번 1 x PBS와 셀 린스.</li>
		<li>녹색 염료 활용 된 이차 항 체 제조 업체의 권장된 농도 따라 어둠 속에서 실 온에서 1 h에 대 한 블로킹 버퍼에 VE cadherin 항 체를 가진 세포를 품 어.</li>
		<li>3 번 1 x PBS와 셀 린스.</li>
		<li>실 온에서 5 분 동안 cyanine 핵 산 바인딩 염료와 핵 얼룩.</li>
		<li>린스 슬라이드 마운트 coverslips 미디어를 탑재 하는 슬라이드를 사용 하 여 슬라이드에 얼굴.</li>
		<li>Confocal 현미경으로 관찰 합니다. 543 nm 레이저와 560-615 nm의 방출 필터를 사용 하 여 α-SMA (Cy3)의 탐지를 위해. 488 nm 레이저와 505-550 nm의 방출 필터를 사용 하 여 VE cadherin의 검출에 대 한. 일반적으로, TGF-β 치료와 치료, 따라 VE cadherin 신호 감소 하 고 α-SMA 신호에서 증가 관찰 될 것 이다.</li>
	</ol>
</li>
</ol>3. 3 차원 콜라겐 젤 수축 시험
<ol><li>콜라겐 젤 수축 시험 전에 72 h에 대 한 MS-1 세포와 함께/없이 TGF-β의 문화를 수행 합니다.</li>
	<li>유형 준비 나 얼음에 콜라겐 젤. 믹스 차가운 콜라겐 솔루션, 집중 10 x 메모리 매체, 0.05 N NaOH, 2.2% NaHCO3및 8: 1에서 200 mM HEPES pH 7.4 포함 된 콜라겐 희석 버퍼: 1의 비율.</li>
	<li>혼합 제어 또는 내 피 성장 세포 TGF-β-취급 정지 (106 셀/200 μ x 1.0) 200 mL와 콜라겐 젤 솔루션의 800 μ. TGF-β에 대 한 치료 샘플 추가 TGF-β2 (1 ng/mL)는 세포 현 탁 액을.</li>
	<li>12 잘 문화 접시의 각 음에 혼합물의 1.0 mL를 추가 하 고 30-60 분 동안 37 ° C에서 인큐베이터에서 공고히 하 허용.</li>
	<li>젤라틴, 후 1.0 mL MEM-α의 10 %FCS, 50 U/mL 페니실린, 및 50 μ g/mL 스 젤을 오버레이. TGF-β에 대 한 치료 샘플 추가 TGF-β2 (1 ng/mL)는 미디어. 참고: TGF-β 처리 샘플에 대 한 젤 및 문화 미디어 TGF-β를 추가 합니다.</li>
	<li>48 h에 대 한 37 ° C에서 부동 젤 품 어.</li>
	<li>스캐너를 사용 하 여 접시의 아래쪽에서 젤의 이미지 스캔 합니다. 또는 젤은 젤 위에 고정 된 거리에서 디지털 카메라를 사용 하 여 이미지를 기록 합니다.</li>
	<li>ImageJ를 사용 하 여 픽셀 수에 따라 젤 표면적 계량. 자유형 선택 또는 관련된 선택 도구를 사용 하 여 젤을 선택 하거나 선택 하는 젤을 사용 하 여 "이미지 | 조정 | 색상 임계값"도구를, 그리고 다음의 젤을 사용 하 여 영역을 결정" 분석 | 측정"명령입니다.</li>
</ol>4. 잠겨 핵 산 기반 미르 억제제 여 miRNA 활동의 억제
<ol><li>MS-1 셀 표준 문화 조건에 유지 합니다.</li>
	<li>Transfect 잠겨 핵 산 (LNA) 미르 억제제, 합성 미르 duplexes 또는 siRNAs (20 또는 50 nM) 제조 업체의 지시에 따라 lipofection 시 약을 사용 하 여. 세부 사항 및 LNA 미르 억제제, 합성 미르 duplexes와 siRNAs의 소스 우리의 이전 보고서26,27에 설명 했다. MS-1 셀, 제조업체의 지시에 따라 표준 transfection 절차 LNA 미르 억제제, 합성 미르 duplexes 또는 siRNAs의 도입을 위해 잘 작동.</li>
	<li>16-48 h 후 TGF-β2 (1 ng/mL의 최종 농도)와 내 피 세포를 자극.</li>
	<li>RNA 식 분석 (양적 RT-PCR 및 RNA 시퀀싱 (RNA-seq) 분석), immunocytochemical 분석 및 기자 시험을 포함 하 여 다양 한 다운스트림 분석을 진행 합니다. 다운스트림 분석 우리의 이전 보고서20,,2627에 설명 되어 있다.</li>
</ol>

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저자는 공개 없다.

그것은 24 h에 대 한 활성화 된 Ras 및 TGF-β 치료에 MS-1 셀, EndMT 유도 TGF-β 혼자21이 짧은 기간 EndMT를 유도 하는 데 실패 하는 동안 보고 되었습니다. 지속적으로, 우리는 TGF-β 실질적으로 유도 EndMT 더 이상 치료 (48-72 h) 후 MS-1 셀20에 관찰. 인간의 배꼽 등 다양 한 내 피 세포에서 TGF-β (2-6 일)와 장기 치료 정 맥 내 피 세포 (HUVEC), 인간의 피부 microvascular 내 피 세포 (HC...

내 피 엽 전환 (EndMT)는 차별화 된 내 피 세포 다양 한 섬유 아 세포와 같은 mesenchymal 세포1의 결과로 분자 변화를 겪 습 하는 과정입니다. EndMT 처음 심장2,3의 개발 하는 동안 내 피 세포 변환으로 설명 했다. 초기 심장 개발, 심장 관 내부 endocardium 및 외부 심근 구성 됩니다. 이 두 개의 레이어 심장 젤리 라고 하는 세포 외 매트릭스의 계층으로 구분 됩니다. 내 피 세포 마커를 획득, 배아 endocardial 셀 중간 엽 세포로 환승, 기본 심장 젤리, 침략 및 것 밸브 및 심장에 대 한 기초를 제공 하는 심장 쿠션의 형성을 촉진 그리고 semilunar 밸브입니다. 또한, EndMT 관상 동맥 혈관, 복 부 대동맥 및 폐 동맥4,,56을 포함 하 여 다른 배아 혈관 시스템에 pericytes 및 혈관 평활 근 세포의 소스를 되도록 제안 되었습니다. 또한, EndMT7돋 아 생리 신생에 연루 이다.
증거를 축적 하는 것은 EndMT도 여러 심혈 관 질환과 다른 질병1,8에 관련 된 것을 제안 했다. EndMT 관련 된 조건이 포함 혈관 석 회화, 동맥 경화 증, 폐 동맥 고혈압, 동굴 같은 기형, 기관 섬유 증, 정 맥 이식 개장, 이식 부전 신장 이식과 암8, 9,10,11,12,13,14,15,,1617, 18. 최근 보고서 설명 여러 분자 EndMT 마커 신장 이식17에서 신장 이식 부전의 진단 및 예 후 예측에 대 한 도구가 될 수 있습니다. EndMT 관련 세포 신호 통로의 변조 표시 되었습니다 심장 섬유 증을 포함 하 여 몇몇 질병 상태를 개량 하 고8,15모델 동물에 정 맥 이식 개장. 따라서, 메커니즘을 이해 기본 EndMT는 EndMT를 대상으로 하는 진단 및 치료 전략을 개발 해야 합니다.
EndMT-셀 접합, 철새 가능성, VE cadherin 같은 내 피 관련 유전자의 downregulation α-부드러운 근육 걸 (α-SMA)를 포함 한 엽 유전자의 upregulation의 손실에 의해 특징입니다. 또한, EndMT 및 상피 엽 전환 (응급), 중간 엽 세포, 상피 세포를 변환 하는 비슷한 과정 변경 된 생산의 발전에 기여할 수 있습니다 다양 한 세포 외 매트릭스 구성 요소와 연결 된 조직의 섬유 증8,19의.
최근, 여러 생체 외에서 연구 EndMT의 EndMT15,20의 분자 메커니즘의 세부 사항을 해명 했습니다. EndMT 변형 성장 인자 (TGF) 등 다양 한 신호 통로 의해 유도-β, Wnt,1단계 고. 그 중 TGF-β 응급 및 EndMT의 유도에서 중추적인 역할을 재생합니다. EndMT에 짧은 노출 부족21나타납니다 동안 노출 EndMT 다양 한 내 피 세포에서 TGF-β에 연장 한다. 우리는 여기 EndMT 유도, 어떤 마일 스벤 1 (MS-1)에서 마우스 췌 microvascular 내 피 세포 받 다 생체 외에서 EndMT TGF-β20에 장기간된 노출 후에 대 한 간단한 프로토콜을 설명 합니다. 이 모델에서 여러 다운스트림 분석 형태 변경, 내 피 마커의 downregulation 엽 마커 및 염증 성 유전자, cytoskeletal의 upregulation 포함 하 여 EndMT의 특징 기능 조사를 수행할 수 있습니다. 재배열, 및 콜라겐 수축 젤.
MicroRNAs (miRNAs)는 22 ~ nt 다양 한 mRNA 대상22,23posttranscriptional 억압을 직접 작은 규제 RNAs. 씨앗 시퀀스 중재 대상 인식, 통해 miRNAs 대상 유전자의 수백을 억제 하 고 세포 분화, 확산, 운동 성 등 다양 한 세포 기능을 조절. 이 구급 대 및 EndMT, 규제에 대 한 경우 이며 또한 몇몇 miRNAs 응급 및 EndMT,2425의 레 귤 레이 터로 보고 되었습니다. 이 검토에서 제시 하는 EndMT 모델 EndMT에서 miRNAs의 역할을 테스트 하려면 미르 변조 절차와 쉽게 결합할 수 있습니다. 현재 검토 MS-1 세포에서 TGF-β-유도 EndMT를 조사 하기 위해 우리의 실험 절차를 요약 하 고 또한 다른 내 피 세포에서 TGF-β에 의해 EndMT 유도의 조건의 비교를 포함 한다.

<table><tbody><tr><td>MS-1 cells</td><td>American Type Culture Collection</td><td>CRL-2279</td><td></td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>32571036</td><td></td></tr><tr><td>TGF-beta2</td><td>R&amp;amp;D</td><td>302-B2-002</td><td></td></tr><tr><td>4 well Lab-Tek II Chamber Slide</td><td>Thermo Fisher Scientific</td><td>154526</td><td></td></tr><tr><td>Y-27632&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>Blocking One</td><td>nacalai tesque</td><td>03953-95</td><td></td></tr><tr><td>phalloidin-tetramethylrhodamine B isothiocyanate</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>TOTO-3 iodide</td><td>Thermo Fisher Scientific</td><td>T3604</td><td></td></tr><tr><td>VE cadherin monoclonal antibody (BV13)</td><td>Thermo Fisher Scientific</td><td>14-1441-82</td><td></td></tr><tr><td>alpha-SMA Cy3 monoclonal antibody (1A4)</td><td>Sigma-Aldrich</td><td>C6198</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Cover slip</td><td>Thermo Fisher Scientific</td><td>174934</td><td></td></tr><tr><td>Collagen solution</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>Collagen dilution buffer</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>LNA miRNA inhibitor</td><td>EXIQON&amp;nbsp;</td><td>miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)</td><td></td></tr><tr><td>synthetic miRNA duplex</td><td>Qiagen&amp;nbsp;</td><td>miScript miRNA Mimic</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778030</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr></tbody></table>

molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling

내 피 세포의 phenotypic가 소성 기초 심장 혈관 시스템 개발, 심장 혈관 질병, 및 기관 섬유 증과 관련 된 다양 한 조건. 이러한 조건에서 차별화 된 내 피 세포는 엽 같은 고기를 획득. 이 프로세스 내 피 엽 전환 (EndMT) 이라고 하 고 내 피 마커의 downregulation, 엽 마커 및 형태학 상 변화의 upregulation에 의해 특징입니다. EndMT 변형 성장 인자 (TGF)를 포함 하 여 여러 신호 통로 의해 유도-β, Wnt, 및 노치, 그리고 gastrulation, 조직 섬유 증에 대 한 비슷한 상피 엽 전환 (응급) 중요 한 분자 기계 장치에 의해 규제 및 암 전이입니다. EndMT의 메커니즘을 이해 하는 것은 EndMT를 대상으로 하는 진단 및 치료 접근을 개발 해야 합니다. 생체 외에서 EndMT의 강력한 유도 일반적인 유전자 식 서명 특성, druggable 분자 메커니즘을 식별 및 변조기 EndMT의 화면을 유용 합니다. 여기, 우리 EndMT의 유도 대 한 생체 외에서 방법을 설명합니다. MS 1 마우스 췌 microvascular 내 피 세포 TGF-β에 장기간된 노출 후 EndMT를 받아야 하 고 upregulation 엽 마커 및 형태학 적 변화 뿐만 아니라 여러 염증 성 발산 및 cytokines의 유도 보여줍니다. 예측에 관한 (미르) 변조 분석을 위한 방법 또한 포함 되어 있습니다. 이 메서드는 기본 EndMT 및 EndMT miRNAs의 기여 하는 메커니즘을 조사 하는 플랫폼을 제공 합니다.

TGF-β는 다양 한 내 피 세포에서 EndMT의 강력한 유도. 24 시간 치료 후 MS-1 세포에서 TGF-β로, F 걸 대 한 얼룩 말라 스트레스 섬유 (그림 1A)20의 개편을 보여 줍니다. Y-27632 바위 억제제와 전처리 말라 개편20의 유도 억제. MS-1 내 피 세포 클래식 조약돌 모양의 형태에서 TGF-β 치료 (그림 1B)에 따라 ?...

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Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

내 피 엽 전환 (EndMT)는 EndMT에 관련 된 세포 신호 경로 조사 하는 데 유용의 생체 외에서 유도 대 한 프로토콜을 설명 합니다. 이 실험 모델에서 EndMT MS-1 내 피 세포에서 TGF-β와 치료에 의해 유발 됩니다.

내 피 엽 전환 성장 인자-β 신호 변환에 의해 유도 된의 분자 분석

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

molecular-analysis-endothelial-mesenchymal-transition-induced

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JoVE Journal

Biology

11.2K 조회수.  Massachusetts Institute of Technology. 내 피 엽 전환 (EndMT)는 EndMT에 관련 된 세포 신호 경로 조사 하는 데 유용의 생체 외에서 유도 대 한 프로토콜을 설명 합니다. 이 실험 모델에서 EndMT MS-1 내 피 세포에서 TGF-β와 치료에 의해 유발 됩니다.

비디오: Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

개발<em&gt; 체외</em&gt; 분석은 중간 엽 세포를 시행 한 상피 - 중간 엽 전환의 수축 기능을 평가하는

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

연구

발생학

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

육종 세포의 중간 엽 상피 전환 유도

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

유방암과 정상 세포에서 TGF β 신호 및 TGF β 유도 된 상피 - 중간 엽 전환 연구

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

암 연구학

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

근접-리깅-분석기 사용 이체 유체 전단 응력 조건 하에서 TGFβ/BMP/SMAD 신호의 시각화 및 정량화

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

생화학

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

종양 특이 내피 세포의 분리 및 문화 확장

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

의학

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

PDMS를 이용한 높은 처리량 견인력 현미경 검사법은 상피-중간엽 전이에 대한 성장 인자-β 변환의 용량 의존적 효과를 밝혀냅니다.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

생체공학

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

전략적 내피 세포 관 형성의 분석 : 감소 세포 외 매트릭스 세포 외 기질과 성장 인자를 비교

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

생물학

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.