Extended Data Fig. 3: Nuclear cathepsin translocation occurs already from 8 hpi, is independent of cathepsin activity and does not result in increased Histone 3 cleavage.
a) Time course of nuclear cathepsin activity. Nuclei and STm were enriched by sequential 50 x g and 8,000 x g centrifugation steps, respectively. RAW264.7 cells were infected with wildtype STm 14028s and treated with DCG04-Bodipy-FLike (5μM) for 4 hours prior to harvesting. Samples were separated by SDS-PAGE and visualised using a fluorescent scanner (Excitation 405 nm/Emission 520 nm), followed by immunoblotting for the soluble cytoplasmic protein GAPDH, the bacterial protein RpoD and Coomassie staining for histones as a loading control for nuclear extracts. L = lysatome, N = nucleome, STm = STm enriched fraction. Experiment was performed once. b) RAW264.7 cells were infected with wildtype STm at MOI 100:1 and harvested at 20 hpi. Whole cell lysates were immunoblotted with the CtsL specific cleavage product of H3 (H3.cs1)16 and histone 3 (H3) as loading control. The experiment was performed in biological duplicate per condition and were analysed in adjacent lanes. c) Nuclear extracts from RAW264.7 cells either mock infected, infected with wildtype STm or heat killed wildtype (STm-HK) for 20 hours. CA-074-Me was present throughout the infection experiment at the indicated concentrations. Nuclear extracts were analysed by immunoblot for CtsB (1:2,000), Lamin A (1:5,000) and histones by Coommassie stain (see Supplementary Information (SI) for second replicate data).
