Fig. 7: Cathepsin-mediated cleavage of histone H3 promotes chromosome segregation.

a Representative confocal images of U2OS cells transfected with indicted plasmids 48 h earlier, n = 3 independent experiments. See Supplementary Fig. 7a for related immunoblots. Scale bars, 10 µm. b Cysteine cathepsin activity (zFR-AFC cleavage) in whole cell lysates of U2OS cells transfected as in (a). Bars, mean + SD, n = 3 independent triplicate experiments. c Activity of rCTSL in the presence of chromatin extract from U2OS cells transfected as in (a) in pH 7,0. Bars, mean + SD, n = 3 independent triplicate experiments. d Quantification of anaphases with chromatin bridges in U2OS cells successfully transfected as in (a) 72 h earlier. Bars, mean + SD, n = 3 independent experiments with >50 anaphases analyzed per sample. e Representative immunoblots of indicated proteins in chromatin extracts of U2OS cells transfected with indicated plasmids as in (a) and incubated for 15 min with rCTSB, rCTSL or 200 nM ALLN at 37 °C, at pH 7.0, as indicated, n = 3 independent experiments. Note that H3-CSTB and H2B are from separate blots prepared in parallel due to the size overlap between a putative H3-CSTB cleavage fragment and H2B. f Quantification of anaphases with segregation errors in U2OS cells successfully transfected with H3-EGFP with wild type histone H3 (WT) or its indicated point mutations 72 h earlier. Bars, mean + SD, n = 4 (WT) or 3 (mutants) independent experiments with >50 anaphases analyzed per sample. g Quantification of interphases with micronuclei in U2OS cells transfected as in (f). Bars, mean + SD, n = 3 independent experiments with >100 interphases analyzed per sample. h Representative immunoblot of H3-EGFP and its cleavage product in chromatin extracts from U2OS cells transfected with indicated H3-EGFP constructs 48 h earlier, n = 3 independent experiments. Chromatin extracts were incubated for 15 min at 37 °C, at pH 7.0 with or without rCTSB and 200 nM ALLN as indicated. P-values were calculated by unpaired, two-tailed Students t-test (b–d) or one-way ANOVA combined with Dunnett’s multiple comparisons (f, g). Source data for b–h are provided as Source Data files.