Figure 6. DMF-dependent assembly of the ripoptosome augments cell death in human Sézary cells.

(A) Specific cell death of T-cells from healthy donors (control) and Sézary patients (n = 5) treated with DMF (50 μM) or vehicle control was assessed. (B) Isolated T-cells from CTCL-positive or -negative donors were stimulated as indicated, stained with AnnexinV-FiTC and 7AAD, and analyzed by flow cytometry. (C) Quantification of AnnexinV-FiTC (AV) and AnnexinV-FiTC/7AAD (AV/7AAD) positive cells normalized to DMF treatment (n = 5). (D) T-cells from Sézary patients (n = 2) were stimulated with vehicle or DMF (50 μM) for 24 h. Complex formation of caspase-8 (C8) and RIPK1 (green) was determined by PLA, Hoechst (red); shown are representative immunofluorescences; scale bar: 10 μm (I), z-stack, merge of C8/RIPK1 and Hoechst; (II) single plane image, merge of C8/RIPK1, Hoechst and Brightfield; (III) z-stack, merge of C8/RIPK1 and Hoechst; (IV) z-stack, C8/RIPK1. (E) Quantification of mean fluorescence intensities acquired from representative images as depicted in (D), (n = 3). (F) Ripoptosome formation in DMF treated Sézary patients. Patient I and II were treated with 480 mg/d DMF for 26 and 10 weeks, respectively. Complex formation of RIPK1 and caspase-8 (C8) was determined by PLA in isolated CD4⁺ T-cells from DMF treated patients and healthy donors (Control I and II) and is depicted in green; nuclear staining with Hoechst is depicted in blue; shown are representative immunofluorescence analyses from z-stacks; scale bar, 10 μm; (left). Mean fluorescence intensities of PLA signals were quantified (n = 4); (right). (G) Depicted are z-stacks from healthy donor (Control II) and DMF treated patient (patient I). Isolated CD4⁺ T-cells were stained for cleaved caspase-3 (cl. C3, green) and Hoechst (blue); scale bar, 10 μm. (A,E,F) Error bars represent standard deviation. Statistics were calculated using Student’s t-test (*p < 0.05; **p < 0.005; ***p < 0.001).