Fig. 3: ALT BIR facilitates efficient viral latency.
From: Oncogenic herpesvirus KSHV triggers hallmarks of alternative lengthening of telomeres
a Schematic overview of Clonogenic assay. b Representative images of Clonogenic assay. See Supplementary Fig. 5 for quantification across biological replicates. c Schematic overview of experiment performed in d. d Depletion of key ALT factors by RNA interference. Cells were treated with siRNA indicated and protein levels were determined at time point of analysis by Western blot with antibodies as indicated. e Quantification of the relative number of KSHV genomes/cell upon depletion of key ALT factors. qPCR was performed against a standard curve of fixed amplicon molecules across conditions to infer copy number. Data presented relative to number determined for scrambled siRNA treatment. Experiment performed in biological duplicate, technical triplicate; Statistical significance tested by ordinary one-way ANOVA using scrambled siRNA condition as control (EA.hy926 ****p < 0.0001, SLK ****p = 0.0014). f Schematic overview of BrdU pulldown experiment (top). Results of experimental quadruplicate measurement by ddPCR upon knockdown of candidate ALT factors. Data expressed as relative KSHV amplicon number normalised to housekeeping control reaction. Statistical significance tested by one-way ANOVA (EA.hy926 nsp = 0.4246, SLK nsp = 0.4777). g Representative micrograph showing virus-associated BIR foci. Images show a maximum intensity Z-projection of confocal images. h Quantification of the occurrence of virus-associated BIR foci (LANA/EdU co-localisation) upon knockdown of key ALT factors. Cells arrested in G2/M were considered. Any residual S-phase cells were discounted from analysis as evident by strong pan-nuclear EdU stain. Data representative of three biological replicates, at least 100 cells were included in the analysis of each condition. Statistical significance was inferred by one-way ANOVA (*p < 0.05, ns, p = 0.069). Error bars in e, f, h represent SEM. i Venn diagram showing overlap of signals considered in analysis depicted in g, h. Percentages were derived from three independent experiments, scrambled control condition only.
