No 1 cover glasses

Cells were grown on no.1 cover glasses (VWR) and infected with SFV4 at a multiplicity of infection (MOI) of 100. At 3 h and 8 h post infection (pi), cells were fixed with 2% glutaraldehyde in 0.1M sodium cacodylate buffer for 30 min at room temperature in the dark. After fixation, cells were washed three times with 0.1M sodium cacodylate buffer, stained with reduced buffered osmium tetroxide and uranyl acetate and processed for flat embedding and ultrathin sectioning as described in [67 (link)]. Images were taken with a Jeol JEM-1400 microscope (80 kV) and a bottom-mounted camera, Gatan Orius SC 1000B.

Cells cultured on 10 mm No. 1 cover glasses (VWR) were incubated with 10 μM BrdU for 24 hours to visualize single-stranded DNA. Cells were treated with 10 μM CB-5083 or 100 μM Mirin for the last 2 and 3 hours, respectively. Ten μM EdU was added 20 minutes prior to fixation to mark S phase. Cells were pre-extracted as above, fixed and the number of BrdU foci imaged using epifluorescence microscopy. For siRNA treatments, cells were treated with siRNA for 48 hours total using RNAiMAX as mentioned. BrdU was added to cells in the last 24 hours before harvesting.

RT112 cells were cultured on 10 mm No. 1 cover glasses (VWR) that had been sterilised with 70% ethanol and rinsed with PBS prior to ionising radiation. After incubation with bacterial supernatants and irradiation, cells were allowed to recover for indicated times prior to permeabilisation with 0.3% Triton X-100. Cells were fixed with ice cold 4% paraformaldehyde and blocked by incubation in 5% BSA, as described previousl y[84 (link)]. After incubation with primary γH2AX (mouse anti-phospho-Histone H2A.X (Ser139) IgG; clone JBW301, Millipore) and secondary (goat anti-mouse IgG, Alexa Fluor 488, ThermoFisher) antibodies, coverslips were mounted on microscopy slides using mounting reagent, Flouromount G, with DAPI (Invitrogen). Fluorescent foci were imaged using a Zeiss 710 confocal microscopy using either a 40X or 63X objective. All microscopy images were analysed with FIJI (ImageJ) software.