Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression
Fig 5
Actin mRNA degradation identifies virally-infected cells experiencing virus-induced host shutoff.
Actin mRNA analysis by qRT-PCR (A,D) or by flow cytometric analysis using PrimeFlow (B,C,E-G), comparing cells with variable infection status. (A) qRT-PCR analysis of beta-actin (Actb) mRNA expression relative to 18s RNA in mock, WT or TMER-TKO infected 3T12 fibroblasts at 18 hpi. (B) PrimeFlow analysis of actin mRNA in 3T12 fibroblasts, either unstained (“No probe”), mock-infected or infected with WT γHV68 or TMER-TKO at 18 hpi. (C) PrimeFlow analysis of actin mRNA and TMER expression in WT γHV68 infected fibroblasts at 18 hpi. (D) qRT-PCR analysis of beta-actin (Actb) mRNA expression relative to 18s RNA in A20, virus-negative cells and A20.γHV68 (HE2.1) cells, untreated or stimulated with TPA for 24 hrs. (E) PrimeFlow analysis of actin mRNA and TMER expression in untreated and stimulated A20.γHV68 cells, with the frequency of TMERhigh actin mRNAlow cells indicated, based on the gated events. (F,G) Actin mRNA analysis by PrimeFlow using either (F) histogram overlays or (G) quantifying frequencies, comparing A20.γHV68 cells that were either untreated or stimulated, further stratified by whether the cells were TMERmid or TMERhigh (using the gating strategy defined in Fig 2C). All flow cytometry data depict single cells, defined by sequential removal of doublets. Data are from two-three independent experiments, with biological replicates within each experiment. Graphs depict the mean ± SEM, with each symbol identifying data from a single replicate. Statistical analysis was done using one-way ANOVA, subjected to Tukey’s multiple comparison test (A, D, G), with statistically significant differences as indicated, ** p<0.01, *** p<0.001, **** p<0.0001.