Figure 6(A) Depletion of Gadd45b expression using lentiviral-transduced shRNAs in latently-infected SCG neurons case cause HSV-1 reactivation. HSV1-GFP-Us11 latently-infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non-silencing shRNA (NS). Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days. Knockdown efficiencies for individual shRNAs in latently-infected SCG neurons were confirmed by RT-qPCR using RNA collected in parallel. Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates. RT-qPCR were quantified from 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (B) smFISH shows that depletion of Gadd45b leads to increased UL36 expression. smFISH was performed 3 days post-lentiviral transduction. smFISH signals were quantified and plotted using ImageJ and Prism. Left: 3 biological replicates, 30 cells were quantified for each replicate. The horizonal lines are mean. Right: 3 biological replicates. The horizonal lines and error bars are mean ± SD. (C) Ectopic expression of Gadd45b-Myc-Flag inhibits viral mRNA expression. Lentivirus-delivered ectopic expression of Gadd45b-Myc-Flag in SCG neurons was confirmed by immunoblotting. Latently-infected SCG neurons were transduced with Gadd45b-Myc-Flag for 3 days and then treated with LY294002 and WAY-150138 to induce reactivation. RNA was collected at the indicated time-points (LY treatment for 18 or 72 hours) and levels of viral UL36 mRNA quantified by RT-qPCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (D) Ectopic expression of Gadd45b-Myc-Flag antagonizes LY294002-induced HSV-1 reactivation. Latently-infected SCG neurons were either transduced with vector control (Ctrl) or Gadd45b-Myc-Flag from lentivirus for 3 days prior to addition of LY294002. Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days. The percentage of wells expressing GFP-positive was scored as the mean ± SEM (n=3 biological replicates). Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates. (E) Ectopic expression of Gadd45b-Myc-Flag slightly inhibited LY294002-induced HSV-1 DNA synthesis. Latent cultures were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138. Total DNA were collected at the indicated times (LY treatment for 18 or 72 hours) post-reactivation in the presence or absence of Gadd45b-Myc-Flag. The relative levels of viral genomic DNA were determined by qPCR using primers for the HSV-1 UL44 gene. Input DNA was normalized by qPCR detection of the rat RPL19 gene. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (F) Ectopic expression of Gadd45b-Myc-Flag suppressed viral ICP4 mRNA expression. Latently-infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138 for 72 hrs. Viral ICP4 mRNA was quantified by qRT-PCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (G) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY-150138 in the presence or absence of Gadd45b-Myc-Flag was quantified by ImageJ (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).