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Fig. 1. Exposure to a series of 10 stresses to UVB at 250 mJ/cm2 or 72 hours of stimulation with TGF-ß1 do not lead to apoptosis. (A) Cytotoxicity at 24 hours after 10 exposures to UVB. Doses of UVB ranged from 0 to 500 mJ/cm² with 2 stresses per day for 5 days. Results are expressed as percentage of cell survival compared to day 0 (d0, 100%) before any stress. Results are given as mean ± s.d. of three independent experiments. Statistical analysis was carried out with Student's t-test. ns, non-significant (P>0.05); ^*, 0.05>P>0.01. (B) No activation of caspase-3 after 10 exposures to UVB at 250 mJ/cm² or 72 hours of stimulation with TGF-ß1. Semi-quantitative confocal microscopy of skin HDFs that were seeded on glass cover slides at 16, 40 or 64 hours after the last of a series of 10 exposures to UVB at 250 mJ/cm² (micrographs 6, 8, 10). Control cells submitted to the same culture conditions but not exposed to UVB were checked (micrographs 5, 7, 9). Cells exposed to etoposide 25 µM for 16 hours were used as positive controls (micrograph 2). Cells incubated for 16 hours in BME were used as negative controls (micrograph 1). At 24 hours after seeding, the activation of caspase-3 was detected using a specific anti-active caspase-3 antibody (green). The nuclei were stained with TO-PRO-3 (blue). No pro-apoptotic effect was found after 72 hours of stimulation with 5 ng/ml of TGF-ß1 (micrograph 4) compared with the controls (micrograph 3). (C) No cleavage of PARP after a series of 10 exposures of skin HDFs to UVB at 250 mJ/cm² or 72 hours of stimulation with TGF-ß1. Skin HDFs were submitted to a series of 10 UVB exposures at 250 mJ/cm². Proteins were extracted at 4, 16, 40 or 64 hours after the last stress. Control cells submitted to the same culture conditions but not exposed to UVB were checked. Cells exposed to cytotoxic concentrations of t-BHP or not were used as respective positive (CTL +) and negative (CTL –) controls. Total cell extracts were analyzed by western blotting with an anti-PARP-1 antibody. The full length PARP-1 protein (116 kDa) and the fragment resulting for PARP cleavage (85 kDa) are indicated. -tubulin protein level was used as a reference. No cleavage of PARP was observed after 72 hours of stimulation with 5 ng/ml TGF-ß1 (TGF) or not (CTL).

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