Supplementary Data Nonerythroid alpha spectrin prevents telomere dysfunction after DNA interstrand...
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Transcript of Supplementary Data Nonerythroid alpha spectrin prevents telomere dysfunction after DNA interstrand...
Supplementary Data
Nonerythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link
damage
Pan Zhang, Utz Herbig, Frederick Coffman and Muriel W.
Lambert
DAPI
αIISp
PNA
8-MOP +UVA
Merge
- +
αIISp
TRF1
- +
DAPI
Merge
αIISp
TRF2
DAPI
Merge
- +
a
c
a b c
a b
Zhang et al.
Figure S1. αIISp associates with telomeres after damage with the ICL agent 8-MOP plus UVA light. Colocalization of αIISp with telomeric DNA, TRF1, and TRF2 in normal cells was examined 16 hr after 8-MOP plus UVA treatment using immunoFISH and staining with anti-αIISp (green), anti-TRF1 (red), or anti-TRF2 (red) antibodies or a Cy3-labeled telomere specific PNA probe (red). Nuclear DNA was counter stained with DAPI (blue). Pictures were taken by z-stack. Only one optical slice is displayed. Magnified images of αIISp colocalization with (a) the PNA probe, (b) TRF1 and (c) TRF2 are shown.
*
αIISp
TRF1
MMC
250
50
- + - +Normal cells FA-A cells
MMC - - + +
*
αIISp
TRF2
250
50
Normal c
ells
FA-A cells
Normal c
ells
FA-A cells
Zhang et al.
Figure S2. Treatment of normal and FA-A cells with MMC has no affect on the levels of TRF1 or TRF2 in these cells. Normal and FA-A cells were treated with MMC (400 nM) and cellular extracts prepared 16 hours after treatment. Levels of TRF1 and TRF2 were determined by western blot. Immunoblots were probed with anti-αIISp, anti-TRF1 or anti-TRF2, antibodies. Topoisomerase (*) was used as a loading control. Molecular weight markers are indicated to the right.
250
50
*
αIISp
TRF1
*
αIISp
TRF2
Normal cells
250
50
αIISp αIISpNt NtsiRNA siRNA
Zhang et al.
Figure S3. Knocking down αIISp in normal cells by siRNA had no affect on levels of TRF1 and TRF2. Normal cells were transfected with αIISp siRNA (100 pM) or Nt siRNA. Levels of αIISp, TRF1 and TRF2 were examined by western blot analysis. Immunoblots were probed with anti-αIISp, anti-TRF1 or anti-TRF2 antibodies. Topoisomerase (*) was used as a loading control. Molecular weight markers are indicated to the right.
*
TRF2TRF1
MMC250
50
- + - +
Normal cells
siRNA Nt αIISp
*
TRF2TRF1
MMC250
50
- + - +
FA-A cells
siRNA Nt µ-calpain
5050
Zhang et al.
Figure S4. Knocking down of αIISp in normal cells and µ-calpain in FA-A cells by siRNA and subsequently treating the cells with MMC (400 nM) had no affect on levels of TRF1 or TRF2. Normal cells were transfected with Nt or αIISp siRNA and FA-A cells were transfected with Nt or µ-calpain siRNA and the cells subsequently treated with MMC (400 nM). Levels of αIISp, TRF1 and TRF2 were examined by western blot analysis. Immunoblots were probed with anti-αIISp, anti-TRF1, and anti-TRF2 antibodies. Topoisomerase (*) was used as a loading control. Molecular weight markers are indicated to the right.
Cells with chromosomal
aberrations
MMC100nM
Nt siRNA
Sister chromatid end to end
fusion
Interchromatid exchanges/
radials Breaks
Total chromosomal
aberrations
- 13.7 ± 5.03 0.03 ± 0.02 0.11 ± 0.04 0.01 ± 0.01 0.043 ± 0.01 0.19 ± 0.08
+ 42.7 ± 7.23 0.12 ± 0.04 0.73 ± 0.09 0.18 ± 0.03 0.37 ± 0.09 1.40 ± 0.24
αIISp siRNA
-84.3 ± 9.29 0.23 ± 0.06 2.55 ± 0.31 0.27 ± 0.06 1.52 ± 0.34 4.57 ± 0.74
+ 100 ± 0 1.08 ± 0.27 7.96 ± 0.30 1.04 ± 0.17 2.02 ± 0.15 12.1 ± 0.85
Normal cells
Chromosome end to end
fusion
Chromosomal aberrations per metaphase a
Table S1. MMC induced chromosomal aberrations in normal cells after knockdown of αIISp by siRNA
a Chromosomal aberrations were counted in 100 metaphases and numbers represent the average from 3 independent experiments.
Zhang et al.
Merge
FANCD2
PNA
Normal cell
- +MMC
FA-A cell
- +
DAPI
0
10
20
30
40
50
Nu
mb
er o
f fo
ci p
er c
ell FANCD2 foci
FANCD2 PNA colocalizedfoci
-normal cells FA-A cells
MMC + - +
Zhang et al.
Figure S5. FANCD2 does not associate with telomeres after DNA ICL damage. (A) Localization of FANCD2 with telomeric DNA in normal lymphoblastoid cells was examined 16 hr after MMC (400 nM) treatment using immunoFISH and staining with anti-FANCD2 (green), or a Cy3-labeled telomere specific PNA probe (red). Nuclear DNA was counterstained with DAPI (blue). Pictures were taken by z-stack. Only one optical slice is displayed. (B) The number of FANCD2 nuclear foci per cell and FANCD2 nuclear foci colocalized with PNA per cell in normal cells before and after MMC treatment was quantitated. 300 cells were counted in each group. Error bars represent S.E.M.
A
B