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. 2023 Dec 22;15(2):e02495-23. doi: 10.1128/mbio.02495-23

Fig 3.

Fig 3

Antiviral Wolbachia strains increase lipid droplet formation in mosquito cells. (A–D) Aag2 cells stably infected with Wolbachia strains wMel, wAlbB, or wPip, or Wolbachia-free cells were mock-infected or DENV-2 infected at MOI 1 for 24 h. (A and C) All cells were stained with BODIPY (493/503) to visualize LDs (green) and DAPI to visualize the cell nuclei (blue). Wolbachia was detected with an α-WSP antibody (red) and 4G2 hybridoma fluid against flavivirus group E antigen for DENV-2 staining (magenta). Scale bars = 15 µm. (B) LD numbers and sizes were analyzed using Fiji software. Data are the mean LD number ± SEM (top) or median LD size ± IQR (bottom) from at least 6 fields of view representing at least 80 cells in total where each data point represents the mean LD number/size from a single field of view. Data are from one experiment, representative of two independent experiments, where each experiment included triplicate biological replicates. Statistical analyses were performed by one-way ANOVA with Tukey’s correction for multiple comparisons (top graph) or Kruskal-Wallis test with Dunn’s correction for multiple comparisons (bottom graph), *P < 0.05, **P < 0.005, ****P < 0.0001. (D) LD numbers were analyzed using Fiji software. Data are the mean LD number ± SEM from at least 6 fields of view representing at least 80 cells in total where each data point represents the mean LD number from a single field of view. Data are from one experiment, representative of two independent experiments, where each experiment included triplicate biological replicates. Statistical analyses were performed by two-way ANOVA with Tukey’s correction for multiple comparisons, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. (E) Ovaries were dissected from female mosquitoes 5–7 days post-emergence and stained with BODIPY (green) and DAPI (blue). Scale bars = 50 µm. All slides were imaged as 3-dimensional z-stacks and 2D images generated by Maximum Intensity Projection (MIP) using Fiji software. Images are from a single representative experiment performed on ovaries from at least three mosquitoes.