Fig. 6. Effects of TGF- on proliferation and apoptosis of HaCaT cells in organotypic co-culture. (A) Proliferation of HaCaT cells in organotypic co-cultures with postmitotic fibroblasts (3x10⁵/ml) grown in DMEM + 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) was evaluated by counting nuclei stained with an anti-BrdU-specific antibody. Bars represent the percentage of BrdU-positive basal keratinocytes identified and counted on cross-sections. Three vision fields per experiment were counted in three independent experiments. (B) Fragmentation of DNA was detected in organotypic co-cultures grown for 12 days in DMEM with 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) by TUNEL labeling (red). Nuclei were counterstained with Hoechst DNA dye (blue). In TGF--treated epithelia at day 12, TUNEL-positive cells are localized in the newly formed stratum granulosum and corneum. Bar, 100 µm. (C) Percentage of HaCaT cells with DNA fragmentation in organotypic co-cultures grown for 12 days in DMEM with 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) was evaluated on cross-sections by counting TUNEL-positive and total number of (bisbenzimide-stained) nuclei. Bars represent percentage of TUNEL-positive cells per epithelium (means ± s.d.). Three vision fields per experiment were counted in three independent experiments. *TUNEL-positive cells at 9 and 12 days are localized in the newly formed stratum granulosum and corneum.

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