Figure 4.

(a) ATM is expressed in HFM’s in vitro. Immunocytochemistry staining showing the nuclear expression of ATM protein (green channel; left image) in cultured primary hair follicle melanocytes (from male scalp, 20-year old). DAPI nuclear stain (blue channel) is co-localised with nuclear ATM staining on the right-hand side image. Scale bars are shown on each image. (b) and (c) ATM expression is increased in HFM after exposure to oxidative stress and can be reduced by antioxidants. HFM were treated with 40 µM H₂O₂ for 1 h in parallel with untreated control cells and cells pre-treated for 1 h with the antioxidants VitE/Q prior to H₂O₂ addition. Levels of ATM protein expression (~ 350 kD) were assessed by Western blot (b). Antioxidant-treated samples were loaded on a second gel and processed in parallel with other samples under identical conditions. GAPDH (~ 36 kD) was used as a loading control. Marker is shown in the first lane of each gel (M). An increase in ATM protein expression could be seen in H₂O₂ treated samples compared to control untreated cells (ns). Levels of ATM could be significantly decreased by preincubation with VitE/Q for 1 h before H₂O₂ treatment (*p < 0.05 versus control, ANOVA; n = 3). Graph in c shows quantification of mean ATM expression (normalised to GAPDH in relative densitometry units) from the Western blot. (d) and (e) Catalase expression is increased in HFMs after exposure to hydrogen peroxide. Representative Western blot showing induction of catalase protein expression (~ 60 kD) in HFM’s after treatment for 1 h with 40 µM hydrogen peroxide. GAPDH (36kD) was used as a loading control. Graph in e shows quantification of mean catalase expression (normalised to GAPDH in relative densitometry units) from Western blots after 40 µM H₂O₂ treatment (n = 3). Unpaired t-test (*p = 0.0113) compared 40 µM H₂O₂ treated cells versus controls.