Supplementary Data

1. Supplementary Data Nonerythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link damage Pan Zhang, Utz Herbig, Frederick Coffman and Muriel W. Lambert

2. Zhang et al. 8-MOP +UVA - + - + - + αIISp αIISp αIISp PNA TRF1 TRF2 c a b Merge Merge Merge DAPI a DAPI DAPI a b c 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.

3. Normal cells FA-A cells Normal cells FA-A cells Normal cells FA-A cells - + - + - - ++ MMC MMC αIISp 250 250 αIISp * * TRF1 TRF2 50 50 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.

4. Zhang et al. Normal cells αIISp Nt siRNA αIISp Nt siRNA 250 250 αIISp αIISp * * TRF2 TRF1 50 50 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.

5. Normal cells FA-A cells µ-calpain Nt αIISp Nt siRNA siRNA - + - + - + - + MMC MMC 250 250 * * 50 TRF1 TRF1 50 TRF2 TRF2 50 50 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.

6. Zhang et al. Table S1. MMC induced chromosomal aberrations in normal cells after knockdown of αIISp by siRNA Chromosomal aberrations per metaphase a Cells with chromosomal aberrations Chromosomeend to end fusion Normal cells MMC 100nM a Chromosomal aberrations were counted in 100 metaphases and numbers represent the average from 3 independent experiments.

7. Zhang et al. A Normal cell FA-A cell - + - + MMC FANCD2 PNA Merge DAPI B 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.