Extended Data Fig. 1: Lack of caspase activity and apoptosis in cells infected with the invasive ShigellaM90T strain. | Nature Microbiology

Extended Data Fig. 1: Lack of caspase activity and apoptosis in cells infected with the invasive ShigellaM90T strain.

From: Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide

Extended Data Fig. 1

a, HeLa cells were either infected with ShigellaM90T (MOI: 30) for the indicated time points or exposed to 100 ng ml−1 Trail or 4 μM STS (3 h). Cytosolic extracts were isolated and analysed by Western blot with the indicated antibodies. PARP cleavage was examined in nuclear extracts. Tubulin served as a loading control. Viability was assessed using trypan blue exclusion assay (n = 3 biologically independent samples). b, Fluorescence microscopy of HeLa cells either infected with ShigellaM90T-dsRed (MOI: 30, 3 h) or exposed to 100 ng ml−1 Trail or 4 μM STS for 3 h and stained with Hoechst (blue). Scale bar, 20 μm. c, Analysis of intracellular bacterial burden by measuring CFU. HeLa cells were infected with the non-invasive ShigellaBS176 or the invasive ShigellaM90T strains and CFU was assessed in cellular lysates. Statistical significance to the corresponding non-infected value was determined by one-way ANOVA followed by Sidak’s post-analysis (n = 3 biologically independent samples). d, HCT cells were infected with the non-invasive ShigellaBS176 or the invasive ShigellaM90T strains. Cytosolic extracts of cells (MOI: 30, 3 h, 100 µg) were treated with recombinant active caspase-8 and caspase-3/-7 activity was measured. Statistical significance to the corresponding unstimulated value was determined by one-way ANOVA followed by Sidak’s post-analysis (n = 3 technical replicates of independent staining). e, HeLa cells were infected with increasing MOIs of ShigellaM90T and treated with 100 ng ml−1 Trail or 4 µM STS. Caspase-3/-7 activity (by Caspase-Glo 3/7 assay) was monitored after 3 h. Statistical significance to the corresponding non-infected value was determined by one-way ANOVA followed by Sidak’s post-analysis (n = 3 biologically independent samples). All experiments are representatives of at least two independent experiments and are presented as the mean ± s.d. **** p ≤ 0.0001; ** p ≤ 0.01; * p ≤ 0.05. Weights in Western blots are in kDa.

Source data

Back to article page