Fig. 5. EGF-receptor signalling is essential for organotypic growth and differentiation of HaCaT cells. (A) Organotypic co-cultures of HaCaT cells with postmitotic fibroblast (3x10⁵/ml) in collagen type-1 gel were grown for 10 days in DMEM as a control (HaCaT), and after addition (every second day) of TGF- (2 ng/ml) or EGF (2 ng/ml) as well as both factors (1 ng each), together with the EGF-receptor blocking antibody C225 (+Ab). For comparison, organotypic co-cultures of normal epidermal keratinocytes were grown for 10 days on collagen gels with 3x10⁵/ml postmitotic fibroblasts (NEK) and with EGF-receptor blocking antibodies (+Ab). (H and E staining; bar, 100 µm). (B) Cytokine and receptor expression in HaCaT epithelia of organotypic co-cultures stimulated for 16 hours with TGF- (2 ng/ml) and EGF, respectively. Expression levels of IL-1, IL-1ß, KGF-R, GM-CSF-R, GM-CSF-Rß, EGF-R and TGF- were determined by semiquantitative RTPCR using GAPDH expression as an internal standard. The RNA expression profile is representative for duplicate determinations of three independent experiments.