Fig. 4. MhAT3F organoids are sensitized to Fas-induced apoptosis. (A-F) Monolayers (2D) or 3D organoids (3D) were treated with the anti-Fas antibody (Jo2) in the presence or absence of cycloheximide, as indicated. (A) Phase-contrast microscopy. Arrows indicate organoids enlarged in inserts. (B) Pooled floating and adherent monolayer cells deposited on microscope slides by cytospin (top) or sections of Matrigel (bottom) were analysed by immunofluorescent staining of active caspase 3. Nuclei are shown in blue (Hoechst 33258), caspase 3 in green (fluorescein isothiocyanate). (C,D) Quantification of caspase-3-positive cells in monolayer cultures (C; 450 cells counted) and in 3D cultures (D; 40 organoids counted). (E) Monolayer cultures treated with TNF with or without actinomycin D, as indicated, were collected and analysed as in C. (F) 3D organoids were treated with TNF with or without actinomycin D and analysed as in D. (G) Cells grown in collagen sandwich were treated as indicated and analysed by phase-contrast microscopy (top) or by immunofluorescent staining of the entire collagen plug with antibody against active caspase 3 (green). Nuclei were stained with Hoechst 33258 (blue). Bars, 20 µm (A,B), 40 µm (G).