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

실험계획

Amplification plot and melting curve analysis: PARP-1, LMNA, b-actin

LMNA 1d-NR 0.3855

LMNA 1d-DMSO 0.53539

LMNA 1d-200 0.48142

LMNA 1d-400 0.38373

LMNA 1d-800 0.72009

LMNA 1d-1000 0.79957

mRNA expression: LMNA exon1 (Normalized with b-actin gene)

1day zebrafish embryos

LMNA 2d-NR 0.34828

LMNA 2d-DMSO 0.62649

LMNA 2d-200 1.40467

LMNA 2d-400 1.24756

LMNA 2d-800 2.74092

LMNA 2d-1000 2.4836

mRNA expression: LMNA exon1 (Normalized with b-actin gene)

2day zebrafish embryos

PARP-1 1d-NR 1.2828

PARP-1 1d-DMSO 1.36417

PARP-1 1d-200 0.90576

PARP-1 1d-400 0.48534

PARP-1 1d-800 0.68389

PARP-1 1d-1000 0.82323

mRNA expression: PARP-1 exon4 (Normalized with b-actin gene)

1day zebrafish embryos

PARP-1 2d-NR 0.96782

PARP-1 2d-DMSO 0.37791

PARP-1 2d-200 1.58276

PARP-1 2d-400 0.99544

PARP-1 2d-800 0.46544

PARP-1 2d-1000 0.90354

2day zebrafish embryos

mRNA expression: PARP-1 exon4 (Normalized with b-actin gene)

1. Slide glass 에 target cell cytospin (600rpm, 5min)2. 4% paraformaldehyed/PBS, 10min, RT 에서 고정3. PBS washing, 5min, 3times4. PBS-T(0.1% Tween20), 15min incubation5. Primary antibody (PARP-1) 1:100 in PBST, 4°c O/N6. PBS-T(1% BSA 첨가 ) washing, 5min, 3times7. Secondary antibody (mouse Green 488) 1:500 in PBST, 2~3hrs Mitotracker Red CMXRos 200nM 처리 8. PBS-T(1% BSA 첨가 ) washing, 5min, 3times9. PBS:DAPI=10,000:1, 10min10. PBS-T(1% BSA 첨가 ) washing, 5min, 2times11. PBS washing, 5min, 1times

Immunofluorescence stain

5d_Bap 100µM

1. cover glass 에 target cell cytospin (600rpm, 5min)2. 4% paraformaldehyed/PBS, 10min, RT 에서 고정3. PBS washing, 5min, 3times4. PBS-T(0.1% Tween20), 15min incubation5. Primary antibody (PARP-1) 1:50 in PBST, 4°c O/N6. PBS-T(1% BSA 첨가 ) washing, 5min, 3times7. Secondary antibody (mouse Green 488) 1:100 in PBST, 2~3hrs Mitotracker Red CMXRos 1:100 (10nM) 처리 8. PBS-T(1% BSA 첨가 ) washing, 5min, 3times9. PBS:DAPI=10,000:1, 10min10. PBS-T(1% BSA 첨가 ) washing, 5min, 2times11. PBS washing, 5min, 1times

Trouble shooting: Mitotracker Red 의 적정 희석배수와 염색시간 찾기 – 10nM, 1nM, 0.1nM, 0.01nM 에서 10 분 , 30 분 염색 testing

SETBP1 mutations in MDS/MPN

Nature Genet-ics

Nature Genet-ics

No. 등록번호 이름 진단명1 18681799 김병조 Atypical CML

2 71148645 김천석3 70769053 박봉심4 70891553 박정희5 70487425 이나리6 16515403 이영옥7 06108625 이재필8 70370093 임점순9 16748652 조광일

10 70891943 조규완11 12166963 김춘자 CMML

12 06465527 한일국13 14328118 박연님14 13936755 하숙자15 06019541 이병기16 70313562 박종금17 70710289 박병동18 11891461 김병옥19 70944733 박종우20 17580835 조규윤21 14832752 정암일22 70147274 고재선23 18998223 김영식24 18922803 양행묵25 18951919 김근호26 19728716 정우진 JMML

27 70796465 민지인28 19995606 박시후29 19090444 김택종 MDS MPN

30 07837331 김재길

Tumor type Number of samplesMDS/MPN Atypical CML 10 CMML 15 JMML 3 MDS/MPN 2Total 30

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

607bp

G2898 (Asp868)

G2898G/A (Asp868Asp/Asn)

Patient No.7 Patient 9 Patient No.10

Patient No.13

C2903 (ser869)

C2903T/C (ser869)

Tumor type Number of samples

Number of Heteroplasmic mutated samples

Percent mutated samples

MDS/MPN 　 Atypical CML 10 3 30

CMML 15 1 6.6

JMML 3

MDS/MPN 2

Total 30