Figure 4. MMP and trypanolysis in control and NHS- or rAPOL1-treated T. brucei.

(a) Effect of TbEndoG RNAi (2 days with Dox) (overnight incubation with NHS or rAPOL1; error bars: s.e.m.; three replicates; n=3; statistical significance: two-way analysis of variance, Sidak post hoc). Right panel: lysosomal swelling and tetramethylrhodamine ethyl ester perchlorate staining in TbEndoG RNAi cells treated with 30% NHS (scale bars, 2 μm; arrow: swollen lysosome). (b) Detection of TbEndoG in T. brucei following 1-h incubation with 30% NHS or 10 μg ml⁻¹ rAPOL1 (Hoechst labels DNA; scale bars, 2 μm); (c–e) apoptotic-like features in the nucleus following 1-h incubation with 30% NHS or 10 μg ml⁻¹ rAPOL1: (c) transmission electron microscopy (scale bars, 200 nm; n, nucleolus; arrows: heterochromatin patches); (d) electrophoretic migration of purified genomic DNA (ethidium bromide staining); (e) terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling of DNA (TUNEL) in control (Ctrl) or TbEndoG RNAi cells; the FITC signal of fluorescent nucleotides transferred at cleaved DNA ends is directly proportional to DNA degradation (arrows: kinetoplast DNA labelling in dividing cells; scale bars, 2 μm; error bars: s.e.m.; three replicates; n=3; statistical significance: two-way analysis of variance, Sidak post hoc). This experiment includes 30% NHS depleted of APOL1 following successive elution on SRA beads (APOL1⁻ NHS), as illustrated in a western blot incubated with anti-APOL1 antibodies.