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Fig. 5. NF-B regulates Fas-induced apoptosis. (A) Monolayer-grown mhAT3F were co-transfected with vectors encoding a truncated rat CD2 and IBAA and treated with the Jo2 anti-Fas antibody. Floating and adherent cells were pooled, deposited on a slide by cytospin and analysed (nuclei are blue, active caspase 3 is green and CD2 is red). (B) Quantification of cells containing active caspase 3 within the subpopulation of transfected cells. (C) Western blot of NF-B p65 subunit in total cell extracts of cells grown in monolayer (2D) or as organoids in Matrigel (3D). Actin serves as a loading control. (D) Monolayer cultures of mhAT3F cells were co-transfected with NF-B/firefly-luciferase and constitutive Renilla-luciferase reporter constructs. 18 hours later, cells were replated and grown either as monolayer cultures (open bars) or 3D organoids (hatched bars) and stimulated by the anti-Fas antibody Jo2 or TNF, as indicated, for 7 hours. Results are shown as means±s.e.m. Bars, 20 µm.

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