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. 2023 Nov 23;31(1):40–52. doi: 10.1038/s41418-023-01237-7

Fig. 7. Loss of CTSB prevents proteins cleavage and suppresses necroptosis in NTD-DmrB cells.

Fig. 7

a CTSB was inactivated by CRISPR/Cas9-mediated knockout in NTD-DmrB cells and CTSB enzyme activity was analyzed in lysates from WT and CTSB-KO cells. ***p < 0.001. b Cells were treated with DMSO or D/Z for 8 h and stained with Hoechst and Sytox Green. Scale bar, 100 μm. c Cells were treated with DMSO or D/Z for 8 h and cell survival was analyzed by CellTiter-Glo assay. ***p < 0.001. Cells were treated for 2 h and cell lysates were analyzed by non-reducing SDS-PAGE (d) or SDD-AGE (e) and probed with a FLAG antibody. f Cell lysates were analyzed by Western blotting with the indicated antibodies. Arrows denote cleaved bands. g Working model. Upon D/Z treatment, NTD-DmrB tetramers form polymers on the lysosomal membrane, leading to LMP and release of active cathepsins into cytosol. Released CTSB cleaves MFN1, MFN2, Lamin A/C, Vimentin and HSP70 to promote mitochondrial fragmentation, nuclear membrane leakage, cytoskeleton disruption and further lysosome permeabilization, eventually resulting in cell death.