The median duration of mitosis for uninfected cells was about 45 min and is indicated by a straight line

The median duration of mitosis for uninfected cells was about 45 min and is indicated by a straight line

The median duration of mitosis for uninfected cells was about 45 min and is indicated by a straight line. For this, propidium iodide (PI) staining was combined with flow cytometric analysis to identify the cellular DNA content. Cell cycle phases were designated according to cellular DNA content. G1 refers to a single set of chromosomes (i.e. DNA content ?=? 1), G2/M to duplicated chromosomes (DNA content ?=? 2), and S refers to replicating chromosomes between the two states (DNA content 1 and 2). Error bars indicate SD.(TIF) ppat.1004162.s003.tif (141K) GUID:?92DA5EF4-700F-4423-9652-EDE414E88371 Figure S4: Internalization occurs in S-phase-arrested cells. (A) HeLa cells arrested MYH9 in S-phase by Adiphenine HCl aphidicolin were infected with AF488-labeled HPV16 PsV for 6 h. Surface-bound PsV were then removed by protease treatment and the remaining cell-associated fluorescence (internalized virus) was measured by flow cytometry. Adiphenine HCl Results are depicted as internalization relative to untreated cells in percent SD. (B) HeLa cells were infected with HPV16-GFP in the presence of aphidicolin, and the drug was replaced at 24 h p.i. by NH4Cl to block acid-activation of viruses. Cells were fixed 48 h after washout and analyzed for GFP expression by flow cytometry. Given are infected cells relative to untreated cells in percent SD.(TIF) ppat.1004162.s004.tif (191K) GUID:?74A294AD-0421-494B-9136-60C9156A9059 Figure S5: Nuclear import occurs in S-phase-arrested cells. (A) To analyze nuclear import of vDNA in the presence of inhibitors, Adiphenine HCl HeLa H2B-mCherry cells arrested in S-phase were infected with recombinant HSV-1-GFP (0.1 PFU/cell). Images were acquired by time-lapse microscopy in 10 min intervals after infection. Indicated is the time after image acquisition started, and after aphidicolin addition (in brackets). (B) HeLa cells treated with Adiphenine HCl aphidicolin or monastrol at indicated concentrations were infected with HSV-1- GFP (0.1 PFU/cell, see also Video S4). Given is the amount of infected cells relative to solvent treated control SD of three independent experiments. (C) To analyze nucleo-cytoplasmic shuttling by cellular importins in S-phase-arrested cells, HeLa IBB-GFP cells were treated overnight with cycloheximide (to prevent IBB-GFP expression) and with aphidicolin. Subsequently cycloheximide was washed out (0 h), and cells were cultivated in the presence or absence of aphidicolin for 10 h to allow IBB-GFP expression and nuclear import. Depicted are microscopic images of a representative cell for each condition. (D) Quantification of nuclear IBB-GFP from (C). The intensity of IBB-GFP within the nucleus was quantified using ImageJ whereby the nuclear area was based on the Hoechst stain. The signals were normalized to cells without aphidicolin, and are depicted as relative nuclear import SD. (E) HeLa cells were transfected with three different siRNAs targeting NUP153 or with control siRNA (AllStarNeg). Cells were infected 48 h post transfection with HSV-1 or HPV16 PsV. Infection was scored at 6 h or 36 h p.i., respectively, by automated microscopy and computational image analysis. Depicted is the infection relative to AllStarNeg control in percent SD.(TIF) ppat.1004162.s005.tif (1.7M) GUID:?A860A2FF-540E-483F-9F26-81F2291775C6 Figure S6: Mitotic indices of RNAi screen. The mitotic state of cells was determined according to nuclear morphologies at the end of the siRNA screen. Depicted is the distribution of the z-scores of mitotic indices for all confirmed genes that enhanced or reduced HPV16 infection (see Table S1). Dashed line indicates the average z-score for all genes.(TIF) ppat.1004162.s006.tif (42K) GUID:?96395561-A85F-4EF6-845D-9ED26F92A07F Figure S7: Characterization of mitoses in relation to HPV16 infection. (A) To examine GFP onset post mitosis in relation to mitosis duration, monastrol-treated Adiphenine HCl HeLa H2B-mCherry live cell infection data (as in bottom panel of Figure 2A) were evaluated as described for Figure 2B and plotted according to GFP onset and mitosis duration. Given is.