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Report
실험계획
1. hTERT cell line에 PAH 노출 후 molecular study (cell count, Viability,
Copy number variation등)
2. Leukemia cell line
- THP-1 cell line 에서 발굴된 Protein {Prelamin-A/C(LMNA), Poly [ADPribose] polymerase 1(PARP-1)}의 mRNA 수준에서 expression study
- Western blotting을 이용한 LMNA, PARP-1 Protein 분석: mitochondria
fraction, cytosolic fraction
- Immunofluorescence stain을 이용하여 PARP-1과 LMNA가 localization
되어 있는 위치 확인
3. In vivo Zebrafish model
- Western blotting을 이용한 Protein 분석: 시약주문 완료
- Protein (LMNA, PARP-1)의 mRNA 수준에서 expression study
4. SETBP1 mutation in MDS/MPN Patients
Amplification plot and melting curve analysis
: PARP-1, LMNA, b-actin
mRNA expression: LMNA exon1 (Normalized with b-actin gene)
1day zebrafish embryos
LMNA
LMNA
LMNA
LMNA
LMNA
LMNA
1d-NR
1d-DMSO
1d-200
1d-400
1d-800
1d-1000
0.3855
0.53539
0.48142
0.38373
0.72009
0.79957
mRNA expression: LMNA exon1 (Normalized with b-actin gene)
2day zebrafish embryos
LMNA
LMNA
LMNA
LMNA
LMNA
LMNA
2d-NR
2d-DMSO
2d-200
2d-400
2d-800
2d-1000
0.34828
0.62649
1.40467
1.24756
2.74092
2.4836
mRNA expression: PARP-1 exon4 (Normalized with b-actin gene)
1day zebrafish embryos
PARP-1
PARP-1
PARP-1
PARP-1
PARP-1
PARP-1
1d-NR
1d-DMSO
1d-200
1d-400
1d-800
1d-1000
1.2828
1.36417
0.90576
0.48534
0.68389
0.82323
mRNA expression: PARP-1 exon4 (Normalized with b-actin gene)
2day zebrafish embryos
PARP-1
PARP-1
PARP-1
PARP-1
PARP-1
PARP-1
2d-NR
2d-DMSO
2d-200
2d-400
2d-800
2d-1000
0.96782
0.37791
1.58276
0.99544
0.46544
0.90354
Immunofluorescence stain
1.
2.
3.
4.
5.
Slide glass에 target cell cytospin (600rpm, 5min)
4% paraformaldehyed/PBS, 10min, RT에서 고정
PBS washing, 5min, 3times
PBS-T(0.1% Tween20), 15min incubation
Primary antibody (PARP-1)
1:100 in PBST, 4°c O/N
6. PBS-T(1% BSA 첨가) washing, 5min, 3times
7. Secondary antibody (mouse Green 488)
1:500 in PBST, 2~3hrs
Mitotracker Red CMXRos 200nM 처리
8. PBS-T(1% BSA 첨가) washing, 5min, 3times
9. PBS:DAPI=10,000:1, 10min
10. PBS-T(1% BSA 첨가) washing, 5min, 2times
11. PBS washing, 5min, 1times
5d_Bap 100µM
1.
2.
3.
4.
5.
cover glass에 target cell cytospin (600rpm, 5min)
4% paraformaldehyed/PBS, 10min, RT에서 고정
PBS washing, 5min, 3times
PBS-T(0.1% Tween20), 15min incubation
Primary antibody (PARP-1)
1:50 in PBST, 4°c O/N
6. PBS-T(1% BSA 첨가) washing, 5min, 3times
7. 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, 3times
9. PBS:DAPI=10,000:1, 10min
10. PBS-T(1% BSA 첨가) washing, 5min, 2times
11. PBS washing, 5min, 1times
 Trouble shooting: Mitotracker Red의 적정 희석배수와 염색시간 찾기 –
10nM, 1nM, 0.1nM, 0.01nM에서 10분, 30분 염색 testing
SETBP1 mutations in MDS/MPN
Nature Genetics
Nature Genetics
No.
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등록번호
18681799
71148645
70769053
70891553
70487425
16515403
06108625
70370093
16748652
70891943
12166963
06465527
14328118
13936755
06019541
70313562
70710289
11891461
70944733
17580835
14832752
70147274
18998223
18922803
18951919
19728716
70796465
19995606
19090444
07837331
이름
김병조
김천석
박봉심
박정희
이나리
이영옥
이재필
임점순
조광일
조규완
김춘자
한일국
박연님
하숙자
이병기
박종금
박병동
김병옥
박종우
조규윤
정암일
고재선
김영식
양행묵
김근호
정우진
민지인
박시후
김택종
김재길
진단명
Atypical CML
CMML
JMML
MDS MPN
Tumor type
MDS/MPN
Atypical CML
CMML
JMML
MDS/MPN
Total
Number of samples
10
15
3
2
30
M
1
2
3
4
5
6
7
8
9
10
11 12 13
14 15
607bp
M
16 17
18 19 20 21
22 23 24 25
26
27 28 29 30
G2898 (Asp868)
G2898G/A (Asp868Asp/Asn)
Patient No.7
Patient 9
Patient No.10
C2903 (ser869)
Patient No.13
C2903T/C (ser869)
Tumor type
Number of s Number of Heteroplasmic mutated
amples
samples
Percent
mutated samples
MDS/MPN
Atypical CML
10
3
30
CMML
15
1
6.6
JMML
3
MDS/MPN
2
Total
30

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