Figure 4(A) smFISH images showing nuclear Gadd45b mRNA in latent (UL36-negative) neurons and nuclear/cytoplasmic Gadd45b mRNA in viral UL36-positive neurons. Latently-infected SCG neurons treated with LY294002 and WAY-150138 for 48 hrs were processed using smFISH to detect Gadd45b (green) and viral UL36 (red) mRNA levels and colocalization. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B) smFISH images processed as in (A) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002. Scale bar, 10 μm. (C) Elevated levels of Gadd45b mRNA in reactivating neurons. Gadd45b signal intensity in HSV-1 latently-infected neurons either untreated (latent) or treated with LY294002 and WAY-150138 for 48 hrs (reactivating) were quantified by ImageJ and plotted. Red line indicates the mean. 3 biological replicates, 25~30 cells were quantified for each replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (D) Quantification of Gadd45b mRNA signal intensity at 48 hrs post treatment with LY294002 or DMSO in uninfected neurons. Red line indicates the mean. 3 biological replicates, 25~30 cells were quantified for each replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns). (E) The percentage of HSV-1 latently-infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY-150138 were scored for either predominantly nuclear or nuclear/cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM. Each purple dot represents a biological replicate experiment. (F) Quantification of nuclear or nuclear/cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO. Each light blue dot represents a biological replicate experiment. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001). (G) Regression analysis showing that the signal intensity of Gadd45b mRNA in reactivating neurons at 48 hrs post treatment with LY294002 and WAY-150138 was inversely related to the signal intensity of viral UL36 mRNA. No significant relationship was detected for ICP27 or UL30 mRNAs. Each dot represents a single reactivating neuron (n=3 biological replicates). Data information: R and R square values were calculated using Correlation analysis by Prism 8. (H) Combined smFISH and immunofluorescence (IF) images showing high GFP-expressing neurons (GFP-positive) or low GFP-expressing (GFP absent) neurons simultaneously probed for Gadd45b mRNA. Reactivation was induced by treatment with LY294002 and WAY-150138 for 60 hrs and processed for simultaneous smFISH and IF staining. Right panel shows quantification (as in G) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY-150138 (n=3 biological replicates). The signal intensity was quantified, plotted, and analysed by ImageJ. Bar, 10 μm. Data information: : R and R square values were calculated using Correlation analysis by Prism 8.