JCS03391 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Repair abilities of CPD and 6-4PP in various human primary fibroblasts used in this study. TIG-120 (normal) (open squares), XP12BE (XP-A) (filled squares), XP1CTA (XP-C) (filled triangles) or XP2BI (XP-G) (filled circles) cells were irradiated with 10 J/m² UV and incubated for the indicated periods. Genomic DNA was isolated and the amounts of CPD (left panel) and 6-4PP (right panel) were determined using an enzyme-linked immunosorbent assay. Each point represents the mean of three experiments and bars indicate the s.d. The repair kinetics of asynchronous MSU-2 cells from Fig. 2C is superimposed in red lines.

• Supplemental Figure 2 -

  Fig. S2. XP3OS/T-n cells are impaired in NER activity and UV-induced H2AX phosphorylation under growth-arrested conditions, and their NER activity is restored by ectopic expression of wild-type XPA. (A) Growth-arrested MSU-2 or XP3OS/T-n cells were exposed to 0 or 10 J/m² UV, incubated for 1 hour and stained with anti-γ-H2AX antibody. (B) MSU-2 or XP3OS/T-n cells growing asynchronously were exposed to 40 J/m² UV, incubated for 0 or 4 hours and stained with anti-6-4PP antibody. (C) XP3OS/T-n cells were transfected with pCMV-Myc-DDB2 or pCMV-Myc-XPA using TransFectin lipid reagent (Bio-Rad). After 1 day of incubation, cells were exposed to 40 J/m² UV, incubated for 4 hours and double-stained with anti-Myc polyclonal antibody (Santa Cruz) and anti-6-4PP antibody.

• Supplemental Figure 3 -

  Fig. S3. Caffeine affects the NER-dependent H2AX phosphorylation. (A) Growth-arrested MSU-2 cells were pretreated with or without 10 mM caffeine for 1 hour before UV exposure (10 J/m²). Cells were further incubated for 1 hour in the presence or absence of caffeine and stained with anti-γ-H2AX antibody. (B) MSU-2 cells growing asynchronously were pretreated with or without 10 mM caffeine for 1 hour and exposed to UV (20 J/m²). After 2 hours of incubation in the presence or absence of caffeine, cells were lysed in NP-40 lysis buffer and analyzed by immunoblotting with anti-p-Chk1 (Ser345) antibody.

• Supplemental Figure 4 -

  Fig. S4. Neither ATM nor DNA-PK is the principal kinase for the NER-dependent H2AX phosphorylation. Growth-arrested AT2KY cells were pretreated with or without 200 μM LY294002 (Sigma) for 30 minutes before the addition of etoposide (final 40 μg/mL) or exposure to UV (10 J/m²). Cells were further incubated for 1 hour in the presence or absence of LY294002 and stained with anti-γ-H2AX antibody.

• Supplemental Figure 5 -

  Fig. S5. Peripheral T-lymphocytes purified from mouse lymph node exhibit H2AX phosphorylation following UV exposure. Lymph nodes were isolated from ddY mice purchased from Sankyo Laboratory Service (Tokyo, Japan). Animal experiments were approved by the Animal Care and Use Committee in Kanazawa University and their guidelines were followed. T lymphocytes were purified by removing B lymphocytes using Dynabeads M-450 sheep anti-mouse IgG (H+L) (Dynal Biotech) and magnetic separation. T lymphocytes were irradiated with 20 J/m² UV, incubated for 1 hour and processed for γ-H2AX staining after fixation. Cells were counterstained with PI and analyzed by flow cytometry. Cell cycle distribution before (left panel) or after UV irradiation (right panel) is shown in A, and histograms of γ-H2AX intensity in UV-irradiated (red line) or sham-irradiated (black line) T lymphocytes are shown in B.