Fig. 3. The 20S proteasome is assembled and functional within Pf-derived EVs.

A Western blot analysis of the different fractions separated by OptiPrep velocity gradient centrifugation. Anti-PSMA1 antibody was used to probe the 20S proteasome complex, and anti-PSMD1 was used to identify the 19S particle. As a positive control, antibodies against the EV markers HSP90 and SR1 were used. This experiment was repeated independently three times with similar results. B Proteasome proteolytic activity measurement of Pf-derived EVs with the fluorescent peptide Suc-LLVY-AMC. As a control, uRBC-derived EVs were used. The data was normalized with respect to background levels of fluorescence in the presence of the proteasome inhibitor MG132. Averaging and statistical analysis were performed on three biological replicates using two tailed t-test assuming unequal variances. Error bars represent SD (standard deviation) (**p = 0.0032). C To validate the integrity and activity of the proteasome complex, uRBC-derived, and Pf-derived EVs were lysed and separated using 4% native-PAGE. The activity of the proteasome complexes was analyzed by incubating the gels with the fluorescent peptide substrate Suc-LLVY-AMC (left panel). Quantifications demonstrates the average of five independent biological replicates subjected to paired two-way t-test analysis using log-transformed measurements (*p = 0.028). Error bars represent SEM. The relative abundance of the complexes was assessed by Western blot analysis of the native gel (right panel, showing a representative gel from three independently repeated experiments with similar results.) using an anti-PSMA1 antibody. Samples of RBC lysate and purified 20S proteasome were used as controls. D Naïve RBCs were incubated with EVs purified from Pf-iRBCs or from uRBC cultures; samples were then lysed and separated using 4% native-PAGE and incubated with the fluorescent peptide (left panel) for activity analysis. Band intensity quantifications of five independent experiments were subjected to averaging and paired t-test analysis (*p = 0.021). Error bars represent SEM. Western blot of the native gel (right panel), using an anti-PSMA1 antibody. Samples of uRBCs and uRBCs treated with EVs derived from naïve RBCs were used as controls. E Denaturing gel of the samples analyzed in D. Anti-PSMA1 was used to probe the 20S proteasome, and anti-PSMD1 was used to probe the 19S complex. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All images are representatives of three independent repeats (see Supplementary Fig. S9). F Parasite growth curve in EV-treated RBCs following incubation with Pf-derived EVs with or without the proteasome inhibitor bortezomib. Average and statistical significance was calculated for at least three independent experiments by comparing percentages (relative to control) between treatments with a two-way ANOVA, accounting for treatment and batch (No EVs n = 3, Pf-derived EVs n = 7, Pf-derived EVs + bortezomib N = 7), **p = 0.0015). Error bars represent SEM. Source data are provided in Supplementary Fig. S7 and in the source data file.