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. 2023 Feb 14;14:828. doi: 10.1038/s41467-023-36446-8

Fig. 1. Characterization of the vitamin K-dependent apoptotic reporter cell line.

Fig. 1

a Schematic diagram of activation of Fas-mediated apoptosis. Fas ligand (FasL) or Fas agonistic antibody activates Fas by forming a trimer and death-inducing signaling complex triggering the extrinsic pathway of apoptosis. CRD cysteine-rich domain, CID calcium-inducing domain, DD death domain, FADD Fas-associated protein with death domain. Fas agonistic antibody was indicated as Y shape in blue on the back of the CRD domains of activated Fas. b Diagram of the chimeric reporter protein FIXgla-Fas with the Gla domain of factor IX fused to the extracellular N-terminus of Fas. c Immunofluorescence confocal microscope imaging of HEK293 cells stably expressing the reporter protein FIXgla-Fas. FIXgla-Fas/HEK293 reporter cells were incubated with 11 µM vitamin K (top) or 5 µM warfarin (bottom) for 12 h. Cells were co-immunostained with FITC-labeled anti-carboxylated FIXgla antibody (green) and APC-labeled anti-HPC4 antibody (red). d VKD apoptosis of FIXgla-Fas/HEK293 reporter cells. The reporter cells were incubated with increasing concentrations of vitamin K for 24 h and the cell viability was determined using cell-counting Kit-8. Data are presented as mean ± SD of five independent experiments (n = 5). e Immunoblotting of the carboxylated (top, probed by anti-FIXgla antibody) and total (bottom, probed by anti-HPC4 antibody) reporter protein FIXgla-Fas after the reporter cells were incubated with 11 µM vitamin K at different time points as indicated. Reporter cells incubated with 5 µM warfarin (W) was used as a control. f Inverted microscope imaging of FIXgla-Fas/HEK293 reporter cells at different time points after treated with 11 µM vitamin K. g Immunoblotting of caspase-dependent apoptosis of FIXgla-Fas reporter cells. Reporter cells were incubated with 5 µM warfarin (W) or 11 µM vitamin K (K) at the indicated time points. Top: Activation of caspase-8 was probed by anti-caspase-8 antibody. Full-length caspase-8 was indicated by an arrowhead, activated caspase-8 was indicated by asterisks. Bottom: total FIXgla-Fas reporter protein expression probed by anti-Fas antibody. Similar results were observed at least three times as shown in Fig. 1f and twice as shown in Fig. 1e, g.