12 mm glass coverslips

Cells were plated on cleaned 12 mm glass coverslips (Electron Microscopy Services, 72230-01) and on the following day fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Following quenching with 50 mM NH₄Cl in PBS, cells were permeabilized and blocked in 2% normal donkey serum (Rockland, 017-000-121) and 0.1% saponin (permeabilization buffer) for 30 min at RT. Primary and secondary antibodies (list of antibodies and dilutions in Supplementary Table 6) were diluted in permeabilization buffer and incubated for 1 h at RT. Finally, cells were washed with 0.1% Triton X-100 in PBS to remove background staining and mounted with mounting medium containing 0.01% Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and 68% glycerol in 200 mM sodium phosphate buffer at pH 7.5 with 0.1 μg/mL DAPI. Coverslips were sealed with nail polish. Cells were imaged on a Leica SP8 SMD confocal laser scanning microscope, equipped with an HC PL APO CS2 63x/1.20 WATER objective. Colocalization analysis was performed using the pearsonr function from the Python package SciPy^{87 (link)}. Individual cells were first saved to separate .tiff files with ImageJ without any modifications, and then processed in a fully automated and unbiased fashion using the pearsonr function.

Cells were seeded at equal densities on 12-mm glass coverslips (Electron Microscopy Sciences, catalog #71887–04) that were placed on a plate. After 24–48 hours, cells were incubated for 30 minutes with EU from a Click-iT RNA Imaging Kit (Invitrogen). After incubation, cells were fixed and processed through the kit protocol. After Click chemistry was utilized to add Alexa Fluor 594 to EU, DAPI was added, and then cells were washed twice with PBS. Cells were mounted on slides with ProLong Glass Antifade Mountant (Invitrogen). Confocal images were taken using a Zeiss LSM 780 in the NCI Microscopy Core Facility.

Cells were seeded at equal densities on 12-mm glass coverslips (Electron Microscopy Sciences, catalog #71887–04) that were placed on a plate. After 24–48 hours, cells were incubated for 30 minutes with OPP from a Click-iT Protein Imaging Kit (Invitrogen). After incubation, cells were fixed and processed through the kit protocol. After Click chemistry was utilized to add Alexa Fluor 594 to EU, DAPI was added, and then cells were washed twice with PBS. Cells were mounted on slides with ProLong Glass Antifade Mountant (Invitrogen). Confocal images were taken using a Zeiss LSM 780.

Cells were seeded at equal densities on 12-mm glass coverslips (Electron Microscopy Sciences, catalog #71887–04) that were placed on a 12-well plate. Coverslips were washed once with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature and washed once again with PBS. 70% ethanol was added to the coverslips and incubated at 4°C for 1 hour. Coverslips were washed with 2X SSC with 10% formamide for 5 minutes, then permeabilized with 0.5% Triton X-100 in 2X SSC for 10 minutes at room temperature. Hybridization buffer (10% dextran sulfate, 1 mg/mL yeast tRNA, 10% formamide, 2X SSC, and RNase-free water) with an RNA FISH oligo labeled with a Cy3 fluorophore (1 μl per 100 μl hybridization reaction) was added to each sample and incubated overnight at 30°C sealed in a plastic bag with a few wet kimwipes. After overnight incubation, coverslips were rinsed once and washed with 2X SSC with 10% formamide once for 15 minutes, then washed again for 30 minutes. Coverslips were stained with 5 μg/ml DAPI in 2X SSC and 10% formamide for 30 minutes at room temperature. After DAPI staining, the coverslips were mounted on glass slides with ProLong Glass Antifade Mountant (Invitrogen). Confocal images were taken using a Zeiss LSM 780. Oligo sequences are provided in the supplementary information (S1 Table).

Cells for immunofluorescence assays were grown and infected on 12-mm glass coverslips (Electron Microscopy Sciences, Hatfield, PA, USA). The cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature followed by permeabilization with 0.5% Triton X-100 for 10 min and screening with antibodies for immunofluorescence microscopy as described previously [35 (link)]. Coverslips were mounted with Prolong Gold Anti-fade reagent with DAPI (4’,6-diamidino-2-phenylindole, Thermo Fisher Scientific). Images were obtained using a Zeiss LSM 700 laser-scanning confocal microscope (Zeiss, Oberkochen, Germany). LSCM was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, which is supported, in part, by funding from NIH-NINDS Center core grant (5P30NS047463).

4 × 10⁵ HEK-293T cells were seeded onto 12-mm glass coverslips (Electron Microscopy Sciences). After 16 to 20 h, 80 µL of 5 µM ON-TARGETplus human UBC9 siRNA SMARTpool (GE Dharmacon, Lafayette, CO, USA) was mixed with 320 µL of media and added to the wells. Non-targeting or GAPDH targeting siRNA (GE Dharmacon) was added to control wells. After 72 h, 200 µL of media containing A. phagocytophilum organisms that had been naturally released from infected RF/6A cells. Additionally, cells from one well of each siRNA treatment were harvested for Western blot analysis to confirm knockdown. At 48 h post-infection, cells were processed for microscopy analyses.