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. 2026 Jan 23;16:1724684. doi: 10.3389/fimmu.2025.1724684

Figure 6.

Panels A and B are bar graphs showing total cell and nuclei numbers at 24 and 72 hours post-infection (hpi) for non-infected (n.i.), wild type (WT), and ΔdotA groups, with statistical significance indicated. Panel C displays microscopy images of cells stained for actin, DNA, Coxiella, and TUNEL, alongside a merged view for n.i., WT, and ΔdotA conditions. Panels D and E present bar graphs indicating apoptosis levels in all cells and within OC, respectively, at 24 and 72 hpi, with statistical annotations.

C. burnetii infection of osteoclasts increases their viability in a DotA dependent manner. (A) Osteoclasts differentiated from bone marrow progenitor cells (Figure 1A) were infected after 2 days of differentiation with C. burnetii WT or ΔdotA at MOI 10. Uninfected osteoclasts (n.i.) were used as control. Cells were stained for TRAP and analyzed by light microscopy. Total cell number was quantified with ImageJ software. (B) Osteoclasts previously seeded on glass coverslips were infected with C. burnetii WT or ΔdotA at MOI 10 on day 2 of differentiation. After 24 or 72 hpi, cells were stained for Coxiella (green - Alexa488), actin (red – Alexa647) and DNA (blue - DAPI) and imaged by fluorescent CLSFM (n=3). The total number of nuclei was quantified with ImageJ software. (C) Similar samples were stained in addition for Coxiella (green - Alexa488), actin (red – Alexa647), DNA (blue - DAPI) and TUNEL (white) and imaged by fluorescent CLSFM. Total number of nuclei and TUNEL+ nuclei were quantified with ImageJ software. Images represent the 72 hpi samples. The scale bar represents 50 µm. (n=3) (D) The percentage of TUNEL+ nuclei among the total number of nuclei was quantified after 24 and 72 hpi. Positive control was measured in parallel to the 24 hpi samples. (E) The number of TUNEL+ nuclei per osteoclast was quantified at 72 hpi. Data are shown as mean ± SD. Circles represent individual microscopical fields of view.