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Fig. 3. Effect of activated Notch on the prostanoid biosynthesis pathway and expression of -actin. (A) Cells were treated for 48 hours with IL-1 (10 ng/ml) or vehicle (control) and conditioned media collected from wild-type (WT CM) or Delta1-Fc-transfected (1-Fc CM) confluent HEK 293 cells. The medium was assayed for secreted PLA₂ and PGE₂. Both parameters were expressed as a percentage of that of control cells (3.5±0.5 pmol/min/µg RNA and 27.9±8.7 pg/ml, respectively). The data represent the mean ± s.e.m. of three independent experiments. IL + 1-Fc CM, IL-1 plus 1-Fc CM versus IL-1 plus WT CM; *, P<0.05; **, P<0.01. (B) Cells were transfected with Notch3 intracellular domain (N3-ICD), Notch1 intracellular domain (N1-ICD) or empty (mock) plasmids and treated for 6, 24 or 48 hours with IL-1 (10 ng/ml) and/or vehicle (control). The levels of PLA₂ and PGE₂ secretion were expressed as a percentage of those of control cells. The basal PLA₂ secretion from mock-transfected cells was 7.3±0.3 and 4±1 pmol/min/µg RNA 24 and 48 hours after transfection, respectively. For N3-ICD-transfected cells, this parameter was 5.7±1.7 and 5.2±1.1 pmol/min/µg RNA and for N1-ICD-transfected cells, 4.8±0.8 and 5.1±1 pmol/min/µg RNA. The basal PGE₂ secretion from mock-transfected cells was 18.2±0.6 pg/ml 48 hours after transfection. For N3-ICD- and N1-ICD-transfected cells, this parameter was 17.8±0.5 pg/ml. The data represent the mean ± s.e. of three to six independent experiments. PLA₂-IIA transcripts were assayed by RT-PCR. The results are expressed as a percentage of the PLA₂-IIA mRNA level of control cells and represent the mean ± s.e. of three independent experiments. *, P<0.05; **, P<0.01; ns, non-significant. (C) 15 µg of total proteins from mock, N3-ICD- or N1-ICD-transfected cells treated for 48 hours with IL-1 (10 ng/ml) and/or vehicle (control) were separated by electrophoresis (10% SDS PAGE). -Actin and GAPDH were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning -actin bands normalized to that of GAPDH and are expressed as a percentage of each control cell. C, control; IL, IL-1; *, P<0.05; ns, non-significant.

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