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[Preprint]. 2023 Mar 20:2023.03.19.533379. [Version 1] doi: 10.1101/2023.03.19.533379

Fig. 4. Increased aggregation of RTN4 is associated with maintenance of LCV integrity.

Fig. 4.

(A) Vacuole integrity of L. pneumophila strains at 2 hr post-infection. BMDMs were challenged with indicated strains, fixed and stained for bacteria before and after permeabilization. The percentages of cytosolic bacteria were calculated. Data displayed as mean ± SEM for three biological replicates. (B) Complementation of ΔsdhAΔsde strain in trans. Shown is aggregation of RTN4 in fixed samples of BMDM challenged with L. pneumophila ΔsdhAΔsde harboring psdeA. Bacteria and RTN4 were probed as described (Materials and Methods), scale bar 10 μm. (C) Vacuole integrity of L. pneumophila strains at 4 hr post-infection. Data shown as mean ± SEM for four biological replicates. At least 70 LCVs were counted per replicate (A, C). Statistical significance was tested using one-way ANOVA with Tukey’s multiple comparisons; *p<0.05,**p<0.01, ***p<0.001 (A, C). (D) A Representative micrograph of RTN4 associated with individual LCVs, scale bar 10 μm. (E) Quantification of RTN4 intensity associated with individual LCVs. Loss of SidJ results in increased association of RTN4 about LCV. Images of individual LCVs were captured and pixel intensities of RTN4 staining about regions of interest were determined. More than 70 LCVs were quantified per experiment and data were pooled from 3 biological replicates (**p<0.01; Mann-Whitney test)