IL-1α drives MK rupture thrombopoiesis by reducing functional and mechanical membrane stability in MKs. (A and B) Fetal liver cells from WT mice were cultured for 7 d with IL-1α or TPO, after which MKs were evaluated using atomic force microscopy by pushing a bead-headed cantilever to measure stiffness (A) and pulling up on a membrane-attached cantilever to measure contractile force (B). Representative force-measurement curve, stiffness, and contractile force against cantilevers were shown. n = 20 measurements. (C and D) Fluorescence recovery after photobleaching (FRAP) analysis. Fetal liver MKs were cultured for 7 d with IL-1α or TPO. Some cells cultured with TPO were also treated with Z-VAD (OMe)-FMK (100 µM) 1 d before the experiments. MKs were stained with Di8-ANEPPS and then photobleached in a region of interest (ROI; red box). Restoration of Di8-ANEPPS intensity reflected membrane fluidity and instability. The left panels show representative snapshots during photobleaching and the corresponding Di8-ANEPPS signals. MKs were divided into mature highly segmented (more than two nuclear segmentations) and not segmented (one or two nuclear segmentations) groups. n = 20 examinations from 5 specimens. *, P < 0.05 versus TPO.