SNX3 regulates phagosomal compaction, proteolytic processing, and intracellular survival of borreliae. (A) Western blot of lysates from macrophages transfected with control siRNA, SNX1-specific siRNA#1, SNX3-specific siRNA#1, or a combination thereof, developed with SNX1- or SNX3-specific antibodies or pan-actin antibody as loading control. Molecular weight (in kilodaltons) is indicated on the left. (B) Evaluation of borreliae compaction in cells treated with control siRNA, SNX1 siRNA#1, SNX3 siRNA#1, or a combination thereof. Values are given as mean ± SEM. For specific values, see Table S1. n = 3 (3 × 30). One-way ANOVA; *, P < 0.05; **, P < 0.01. (C–J) SNX3 regulates proteolytic processing within borreliae-containing phagosomes. (C–H) Confocal micrographs of macrophages incubated with DQ-BSA (C and D), to visualize proteolysis, with internalized GFP-expressing borreliae (E and F), with merges (G and H). Macrophages were treated with control siRNA (C, E, and G) or SNX3-specific siRNA (D, F, and H). Scale bars: 10 µm, and 1 µm for insets. (I and J) Evaluation of DQ-BSA–based fluorescence intensities at macrophage phagosomes containing (I) or not containing (J) borreliae. Fluorescence intensities are indicated on the y axis. Macrophages were treated with control siRNA or two individual SNX3-specific siRNAs, as indicated. n = 3, with DQ-BSA signals evaluated in 3 × 30 cells for values in I and 3 × 30 cells for values in J. Values are given as mean ± SEM. For specific values, see Table S1. One-way ANOVA; *, P < 0.05; **, P < 0.01. (K) SNX3 regulates intracellular survival of borreliae. Statistical evaluation of intracellular survival of borreliae in macrophages treated with SNX3-specific siRNA or control siRNA. Graphs show growth curves of borreliae recultured from respective macrophage cell lysates. Number of recultured borreliae in growth medium (104/ml) is indicated on the y axis, and days after start of the experiment are indicated on the x axis. n = 3, two-tailed Student’s t test; *, P < 0.05.
