Particles for HSVEdC that are positive for both signals are in the upper right quadrant and coded yellow. in materials and methods. We used a customised Image J plugin based on the find maxima protocol accompanied by pixel quantification in defined ROIs for each channel (top panel VP5, red; bottom panel EdC, green). Particles were scored positive by being not only above ROI normalised background threshold, but being at least 1 SD above that value. Frequency distributions of signal intensities for each channel of individual VP5 positive particles were quantitated with bin width automatically selected using the Friedman-Diaconis criteria for interquartile-ranges [74, 75]. The same bin width was used for both channels aligning means for both channels for ease of comparison of distributions. The raw mean and SD together with the coefficient of variance are reported TAS4464 on the right-hand side, with fitted means and SDs reported on the left hand side after Gaussian distributions were fitted to each channel frequency data using Image J curve fitter.(TIF) ppat.1006721.s003.tif (1.1M) GUID:?9EA77AF8-75F6-4184-99A8-E11E867B42F0 S4 Fig: Scatter plot analysis of VP5 and genome signals for individual particles. The same analysis used for overall frequency distributions of VP5 and DNA of the total particles (S3 Fig) was used to generate scatter plots where each dot represents an individual particle and its TAS4464 score for VP5 (Y-axis) and EdC (x-axis). The threshold for scoring positive is indicated by the solid lines in red or green. Note that thresholds are calculated with regard to individual images and background for data in each field and may be marginally different. Particles for HSVEdC that are positive for both signals are in the upper right quadrant and coded yellow. Particles which are VP5 positive and below threshold in green are coded red. For HSV w/t essentially no VP5 positive particle exhibited any significant green signal.(TIF) ppat.1006721.s004.tif (342K) GUID:?F86144B6-D1BD-4722-A0D1-D6E867535D79 S5 Fig: HSVEdC genomes are only detectable after adsorbtion onto a solid support. (a) HSV-1EdC virus particles were adsorbed onto borosilicate coverslips, detected by cycloaddition and immunofluorescence and analysed as described for Fig 4. (b) HSV-1EdC was examined by cycloaddition in solution prior to adsorption on coverslips. The reaction was then blocked by addition of 1 1 mM EDTA to chelate the copper catalyst and the sample then adsorbed to borosilicate coverslips, stained for VP5 and analysed by ImageJ. Graphs show quantified data of particles under each condition for VP5 and EdC. (c) HSV-1EdC was adsorbed to borosilicate coverslips as above and then heated to 70C for 2 min before fixation and detection by cycloaddition and immunofluorescence for VP5. VP5, red; EdC, green (scale bar 10 m).(TIF) ppat.1006721.s005.tif (1.9M) GUID:?605EDC3F-22C3-4AA1-9ED6-8E41F012C9CC S6 Fig: Control experiments on entry and uncoating of HSV-1EdC genomes. RPE-1 cells were infected with HSV-1EdC and fixed at 2 hpi for detection by cycloaddition and immunofluorescence for VP5 (scale bar 10 m). Experimental variations from the normal process were as follows: (i) During the cycloaddition, Cu(I) was omitted from the reaction mixture; (ii) the virus inoculum was treated with clinical grade neutralizing antibody IVIg (100 mg/ml) for 0.5 hr at room temperature prior to infection; (iii) the inoculum was treated with DNase I (500 U/ml) for 0.5 h at 10C prior to infection; (iv) cells were infected with a mock inoculum which consisted TAS4464 of concentrated supernatant from uninfected RPE-1 cells pulsed and prepared as for an infected HSV-1EdC stock.(TIF) ppat.1006721.s006.tif (2.2M) GUID:?E9286D67-6DA3-4488-826F-88F20D7545CC S7 Fig: Video of iso-rendered 3D-SIM data of HSVEdC infected cell. 3D-SIM data of a representative RPE-1 cell infected with HSV-1EdC (moi 20) at 0.5 hpi, iso-rendered in Huygens analysis software as described in materials and methods. Channels have been rendered partially transparent for ease of inspection of features as discussed in the text Red channel, VP5; Green channel, EdC; blue channel, DAPI.(AVI) ppat.1006721.s007.avi (52M) GUID:?65FD811D-D602-4D22-A5B6-95D231BF1DF3 S8 Fig: Control experiments on effects of drug treatments. (a) Representative images of the localisation of ICP4 in cells infected with HSV-1EdC (moi 10) and either untreated or incubated in the presence of ActD (5 g/ml), CHX (100 g/ml), or ACV (500 M), PAA (400 g/ml), as indicated. Cells were fixed (3 hr post infection) and processed for immunofluorescence of ICP4. Scale bar 10 m. (b) Uninfected cells were treated with MG132 (10 M), nocodazole (2 M), or Leptomycin B (20 nM) for 1.5 Rabbit polyclonal to Wee1 hr, as during analysis of infection, then fixed and processed for immunofluorescence for PML, -tubulin, or cyclin B1 respectively (red channel). Cells were counterstained with DAPI..
Particles for HSVEdC that are positive for both signals are in the upper right quadrant and coded yellow