Mbv induces multinucleation of bMϕ.

(A) Fluorescence confocal images of bMϕ infected with Mtb-RFP, Mbv-RFP or PFA-killed-Mbv-RFP for 24 h. Non-infected cells (NI) were used as a control. The bacteria are visualized in red, the cells-actin cytoskeleton is in white (phalloidin-488) and cell nuclei (DAPI) in cyan. The white square represents a region of interest magnified below each image. Scale bar, 40 μm. (B) Quantification of the percentage of MNGCs in bMϕ for each condition. Data are representative of two independent biological repeats, each carried out in duplicate. (C) GM-CSF-bMϕ or -hMϕ were infected with Mbv-RFP (red) for 24 h. PFA-fixed infected cells were stained with phalloidin-Alexa Fluor 488 (actin, green). DAPI (blue) was used to stain nuclei. Images presented above were acquired using a confocal microscope. Images were analysed using Harmony software (PerkinElmer). Actin stain was used to mask the cell bodies and determine the number and the area of the cells detected. DAPI staining was used to segment and count the number of nuclei in each cell. Cells containing 2 or more nuclei were considered. For each cell represented by a grey dot, values were plotted as nuclei number as a function of the cell’s area (μm2). Scale bar, 50 μm (D) Electron microscopy image of Mbv-induced bovine MNGCs containing three or six distinct nuclei (red arrows). Scale bar, 10 μm.

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