Fig. 8: Multiplexing reveals that RNAP II phosphorylation fluctuates during HSV-1 infection.

a Distributions of mean nuclear intensities of total RNAP II, RNAP II S5P, RNAP II S2P, and HCFC1 in uninfected and HSV-1-infected HeLa cells from four individual replicate wells in the smFISH + 4i experiment. 0.1–99.9^th percentiles of marker intensities are shown in the density plots. Inset: cell counts per time point. b ICP27, ICP8, total RNAP II, RNAP II S5P, RNAP II S2P, and HCFC1 staining of HSV-1-infected HeLa cells at 12 hpi. Arrows, infected cells containing viral replication compartments (RCs) with RNAP II, RNAP II S5P, and HCFC1 co-localized. Nucleus and cell outlines as in Fig. 1a. Scale bar, 25 μm. c Mean nuclear intensities of total RNAP II, RNAP II S5P, RNAP II S2P, and HCFC1 in uninfected and infected HeLa cells at 12 hpi. Infected cells without RCs were clustered into four classes (I–IV) based on their mean intensity of ICP27 and ICP4. Class V represents infected cells that contain at least one ICP8-positive RC. Data are from four individual replicate wells in the smFISH + 4i experiment, and cell counts are indicated next to plots. Boxplots definitions as in Fig. 1c. Outliers are omitted for clarity. d Single-cell mean intensity of ICP27 and ICP4 in classes I–V of infected HeLa cells, at 12 hpi. e Single-cell mean nuclear intensity of RNAP II S5P or S2P as a function of mean intensity of ICP27 or ICP8 in infected HeLa cells at 3–12 hpi. Classes as in c. f UMAP of infected HeLa cells (as in Fig. 3d) colored by classes as in c. Cells at 1.5 hpi were not included in the clustering and are colored gray. g UMAP of infected HeLa cells (as in Fig. 3d) colored by ICP8 sum intensity, Haralick sum entropy (Kernel size: 10) texture of RNAP II in nucleus, Haralick correlation (Kernel size: 2) texture of HCFC1 in nucleus, and mean nuclear intensities of RNAP II S5P and S2P. Scale bar: lower limit is <0.5^th and upper limit is >99.5^th percentile of the values. See also Supplementary Fig. 15.