Fig 2: ICP27 directly interacts with CPSF and the mRNA 3′ processing machinery.a A network plot of the top ICP27-associated host factors. The purple dots are factors with known functions in mRNA export and the blue dots are mRNA 3′ processing factors. The plot was based on STRING. b Cells infected with WT HSV-1 (KOS) were harvested at different time points post-infection and subjected to immunoprecipitation with an anti-ICP27 antibody. Input and IP samples were analyzed by western blotting. c In vitro pulldown assays with GST-ICP27 and recombinant Fip1, CPSF73, and CstF complex. The input and pulldown samples were analyzed by western blotting. d Top panel: a diagram of known domains in ICP27 protein and the regions deleted in the mutant ICP27 proteins are marked below. Lower panel: WT or various mutant Flag-tagged ICP27 were expressed in cells via transient transfection and IP was performed with anti-Flag antibody. The input and IP samples were analyzed by western blotting. Source data are provided as a Source Data file.

Fig 3: JTE-607 leads to DNA damage and S phase crisis.a Representative flow cytometry data of γH2A.X expression levels in U937/iFIP1 cells in Dox− or Dox+ conditions after JTE-607 treatment for 24 h. b Fold change of γH2A.X signals in U937/iFIP1-A1 cells with or without FIP1 induction after JTE-607 or DMSO treatment. P value is based on the Chi-squared test. Fold change is based on JTE-607-treated vs. DMSO-treated samples in Dox− and Dox+ conditions, respectively. c Cell cycle analysis using U937/iFIP1-A1 cells. Data are based on 24 h post JTE-607 treatment. d Percent of cells in different cell cycle phases as based on data in c. e Ratio of cells in the 1st half S phase to the 2nd half S phase after JTE-607 treatment in U937/iFIP1 cells in Dox+ vs. Dox− conditions (n = 2). P value (t-test) for significance of difference is indicated. f Synergy scores for JTE-607 with another indicated compound in U937/iFIP1 cells in Dox+ or Dox− conditions. Synergy scores were calculated by the SynergyFinder program using the Bliss method. The ‘+/− value’ indicates 95% confidence interval (two replicates). The function of each compound is indicated, namely, TOPIi, TOPIIi, and ATRi. g Representative synergy score plots generated by the SynergyFinder program for JTE-607 in combination with BAY 1895344 in U937/iFIP1-A1 cells in Dox− (left) or Dox+ (right) conditions. h As in f, except that synergy analysis was carried out in U937 cells (two replicates). i A model summarizing mode of action of JTE-607 (CPAi). Alternative polyadenylation and transcriptional readthrough lead to transcriptomic disturbance, the extent of which correlates with the CPA activity of a cell. Transcriptional readthrough also leads to S phase crisis, due likely to transcription-replication conflict, and then DNA damage, which is exacerbated by a high cell proliferation rate. CPAi causes cell death through both transcriptomic disturbance and DNA damage. Source data are provided as a Source Data file.

Fig 4: FIP1 overexpression leads to global activation of CPA activity.a Schematic of a lentiviral vector containing an inducible FIP1 (top) and Western blot (bottom) showing induction of FIP1 in HEK293T cells by 2 μg/mL Dox for one or two days. b Scatter plot comparing 3′UTR isoform abundance difference between HEK293T cells with FIP1 induction (Dox+, 24 h) and those without (Dox−). Each dot represents a gene with two selected 3′UTR isoforms (as shown in Fig. 5a). c As in b, except that IPA isoforms are analyzed. See Fig. 5f for IPA and TPA isoform definitions. d Schematic of reporter assay to examine APA regulation by FIP1 overexpression (OE) using pTRE-FIP1-IRES-BFP. e Bar graph indicating the level of pPAS usage in HEK293T cells with the blue fluorescent signal tracking FIP1 expression level. Cells showing no, low, or high blue signals were analyzed for pPAS usage, as indicated by log2(R/G). Error bars are standard deviation based on three biological replicates. P values (t-test) for significance of difference between comparing groups are indicated. f Inhibition of pPAS usage by JTE-607 in cells with different levels of FIP1 OE. Linear regression formula and R2 values are shown for the cell groups with no or high BFP signals. Error bars are standard deviation of three biological replicates. P value (t-test) for significance of difference in Δlog2(R/G)(10 μM JTE-607 vs. DMSO) between cell groups are indicated. g Schematic of the cell competition assay, in which HepG2 or HeLa cells with inducible FIP1 OE (named HepG2/iFIP1 and HeLa/iFIP1, respectively) are subject to JTE-607 treatment, followed by flow cytometry analysis to examine green or blue fluorescent signals of surviving cells. h Histograms showing distribution of fluorescence signals of HepG2 cells after DMSO (top) or JTE-607 (bottom) treatment. i Bar graph showing ratio of number of cells with low (bottom 1/3) to high fluorescent signals (top 1/3) in HepG2 cells treated with 1 μM JTE-607. Error bars are standard deviation of three biological replicates. P (t-test) for significance of difference is indicated. j As in i, except that HeLa/iFIP1 cell data are shown with 50 μM JTE-607. Source data are provided as a Source Data file.

Fig 5: FIP1 overexpression leads to greater cell sensitivity to JTE-607.a Western blot showing induction of FIP1 in two U937/iFIP1 clones (A1 and B12). b IC50 analysis of U937/iFIP1-A1 and -B12 cells treated with JTE-607. Error bars are standard deviation (n = 3). c Histograms showing distribution of fluorescence signals of U937/iFIP1-A1 after DMSO (left) or JTE-607 (right) treatment. d Bar graph showing percent of U937/iFIP1-A1 cells with different green fluorescence levels after the cell competition assay with 10 μM JTE-607 (n = 2). P is based on t-test. e Schematic showing transcriptome analysis of U937/iFIP1 cells with or without FIP1 induction (Dox+ or Dox−, respectively) followed by JTE-607 treatment. f Gene expression changes after FIP1 induction for 48 h. The number of genes with significant expression changes (BH-adjusted P < 0.05, Fisher’s exact test; fold change ≥1.2 or ≥2) are indicated. A total of 13,056 genes were analyzed. g 3′UTR shortening in U937/iFIP1 cells after FIP1 induction. See Fig. 8b for description of the plot. h Comparison of JTE-607-elicited gene expression changes in U937/iFIP1 cells in Dox+ vs. Dox− conditions. Genes with significant expression changes in both conditions (BH-adjusted P < 0.05, Fisher’s exact test; fold change ≥1.2) are colored with red (both upregulated) or blue (both downregulated). Genes in black are significant in only one condition and genes in gray are not significant in either condition. A total of 11,311 genes were analyzed. Pearson correlation coefficient (r) is indicated. i UpSet plot showing numbers of genes with significant JTE-607-elicited expression changes (BH-adjusted P < 0.05, Fisher’s exact test; fold change ≥2) in U937/iFIP1 cells in Dox+ and/or Dox− conditions. Gray bars indicate different directions of regulation between the two conditions. The ratio of number of downregulated genes to that of upregulated genes is indicated. j As in i, except that JTE-607-elicited 3′UTR APA events are compared between U937/iFIP1 cells in Dox+ vs. Dox− conditions. k qRT-PCR analysis showing JTE-607-elicited transcriptional readthrough of gene HNRNPD in U937/iFIP1 cells with or without FIP1 induction. chrRNA signal in readthrough region was normalized to that in gene body. Error bars are standard deviation (n = 3). P values (t-test, one-tailed) for significance of difference in readthrough are indicated. Source data are provided as a Source Data file.