1. hTERT cell line 에 PAH 에에 에 molecular study (cell count, Viability, Copy number variation 에 ) 2. Leukemia cell line - THP-1 cell line 에에 에에에 Protein {Prelamin-A/ C(LMNA), Poly [ADP-ribose] polymerase 1(PARP-1)} 에 mRNA 에에에에 expression study - Western blotting 에 에에에 LMNA, PARP-1 Protein 에에 : mitochondria fraction, cytosolic fraction - Immunofluorescence stain 에 에에에에 PARP-1 에 LMNA 에 localization 에에 에에 에에 에에 3. In vivo Zebrafish model - Western blotting 에 에에에 Protein 에에 : 에에에에 에에 - Protein (LMNA, PARP-1) 에 mRNA 에에에에 expression study 4. SETBP1 mutation in MDS/MPN Patients Heteroplasmic mutation 에에에 sample TA cloning- 에에에에 에에 5. Zebrafish aging study- sequencing 에에 에에에에

hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

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실험계획. hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 ) Leukemia cell line - THP-1 cell line 에서 발굴된 Protein { Prelamin -A/C(LMNA), Poly [ADP-ribose] polymerase 1(PARP-1)} 의 mRNA 수준에서 expression study - PowerPoint PPT Presentation

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1. hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viabil-ity, Copy number variation 등 )

2. Leukemia cell line - THP-1 cell line 에서 발굴된 Protein {Prelamin-A/C(LMNA), Poly

[ADP-ribose] polymerase 1(PARP-1)} 의 mRNA 수준에서 expres-sion study

- Western blotting 을 이용한 LMNA, PARP-1 Protein 분석 : mito-chondria fraction, cytosolic fraction

- Immunofluorescence stain 을 이용하여 PARP-1 과 LMNA 가 local-ization 되어 있는 위치 확인

3. In vivo Zebrafish model - Western blotting 을 이용한 Protein 분석 : 시약주문 완료 - Protein (LMNA, PARP-1) 의 mRNA 수준에서 expression study

4. SETBP1 mutation in MDS/MPN Patients Heteroplasmic mutation 발견된 sample TA cloning- 시약주문 완료5. Zebrafish aging study- sequencing 완료

실험계획

Page 2: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

1. Mitotracker Red CMXRos 1:100 (10nM) 처리 2. PBS-T(1% BSA 첨가 ) washing, 5min, 3times3. PBS:DAPI=10,000:1, 10min4. PBS-T(1% BSA 첨가 ) washing, 5min, 2times5. PBS washing, 5min, 1times

Immunofluorescence stain

Page 3: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

Mitotacker Red Testing

0.01nM-30min 0. 1nM-30min

1nM-30min 10nM-30min

Page 4: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

다음 실험 Plan

-Hela cell 로 testing 슬라이드 글라스에 키워 놓음

-THP-1 cell: Matrigel 을 이용하여 고정시킨 후 실험 진행

Page 5: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

SETBP1 mutations in MDS/MPN

Page 6: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

G2898G/A (Asp868Asp/Asn)

Patient No.10

G2898 (Asp868)G2898A (Asp868Asn)

Page 7: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

Patient No.13C2903T/C (ser869)

Page 8: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

Aging related-zebrafish study

Page 9: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

Control region PCR

Page 10: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

No. Age Tissue Mutaions1 6m-1 Brain2 Kidney3 Muscle4 6m-2 Brain5 Kidney6 Muscle7 6m-3 Brain8 Kidney9 Muscle10 6m-4 Brain11 Kidney12 Muscle13 1y-1 Brain A339G C582T C635T14 Kidney A339G C582T C635T15 Muscle A339G C582T C635T16 1y-2 Brain A339G C582T C635T17 Kidney A339G C582T C635T18 Muscle A339G C582T C635T19 1y-3 Brain A339G C582T C635T20 Kidney21 Muscle A339G C582T C635T22 1y-4 Brain A339G C582T C635T23 Muscle A339G C582T C635T24 2y-1 Brain A339G C582T C635T25 Kidney A339G C582T C635T26 Muscle A339G C582T C635T27 2y-2 Brain A339G C582T C635T28 Kidney A339G C582T C635T29 Muscle30 2y-3 Brain31 Kidney32 Muscle A339G C582T C635T33 2y-4 Brain A339G C582T C635T34 Kidney A339G C582T C635T35 　 Muscle A339G C582T C635T

Page 11: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

A339GA339

Page 12: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

C582TC582

Page 13: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

C635TC635

Page 14: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

0.6%

Mitotracker RedMeasurement of early apoptosis

Normal

DMSO

Benzopyrene

Pentacene

Fluoranthene

Pyrene

Page 15: hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 )

Normal

DMSO

Benzopyrene

Pentacene

Fluoranthene

Pyrene

Mitotracker GreenMeasurement of Mitochondrial Mass

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A 198 115 90.5 123 IP: HA WCE +++ Hdm2 HA-Ub FLAG-hTERT MG132 - ++ +++ -- + WB: FLAG WB: Hdm2 WB: FLAG hTERT -(Ub)n Supplementary Figure S1. Oh et al

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