15 mm glass coverslips

RAW 264.7 cells were cultured on 15-mm glass coverslips (Thermo Scientific, Waltham, MA, USA) in 24-well plates and infected with the WT strain or the Δ27735 mutant at an MOI of 200, as described above. At 4 and 24 h pi, the cells were washed twice with PBS and fixed overnight in 4% (w/v) paraformaldehyde at 4 °C. Fluorescence staining and a Brucella co-localization assay with lysosomes were performed as described in our previous report [11 (link)]. Rabbit anti-Brucella polyclonal antibody (1:500 dilution) was used to track intracellular bacteria. Rat LAMP-1 (lysosome associated membrane protein 1) monoclonal antibody (1:1000 dilution; Abcam, Cambridge, UK) was used to track the lysosomes. Goat anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 (Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies at dilutions of 1:1000. The cells were observed under laser scanning confocal microscope (Nikon D-Eclipse C1, Tokyo, Japan) with 100× oil immersion objective. Images were saved in TIFF format and imported to Adobe Photoshop CS4 (Adobe Systems Incorporated, San Jose, CA, USA), where they were merged using RGB format. To determine the percentage of bacteria positive for the lysosome marker LAMP-1, 100 intracellular bacteria were counted randomly. Assays were performed in triplicate.

Generation of wild-type, Fuzzy^−/− and ArhGap35^D34/D34 MEFs was previously described (Seo et al., 2011 (link); Stewart et al., 2016 (link)). MEFs were plated on 15 mm glass coverslips (Thermo Fisher Scientific) coated with rat Collagen I (Life Technologies) at 10⁵ cells per well in 12-well plates (Sarstedt). DMEM/F12 growth medium was supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 1% penicillin/streptomycin (all from Wisent). After 24 h, cells were starved in serum-free medium for another 24 h to induce ciliogenesis and then treated with actin polymerization inhibitors for 8 h. Fasudil was diluted to 50 µM in PBS, Y27632 diluted to 1 µM, and cytochalasin D to 0.5 µM in DMSO (all inhibitors from Sigma-Aldrich). Inhibitor concentrations were optimized to ensure high cell viability using Cell Counting Kit - 8 reagent (Sigma-Aldrich) according to manufacturer recommendations. At the end of the incubation period, cells were washed with DMEM and fixed with 4% paraformaldehyde in PBS (PFA/PBS) for 15 min after four 3-min washes with PBS. For colocalization experiments, both Fuzzy^−/− and ArhGap35^D34/D34 MEFs were grown under similar conditions. Human embryonic kidney HEK293T cells were grown in 100 mm Petri dishes in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin until ready for transfection.