Journal: Cells

Article Title: Phosphoproteome Remodeling upon CDK1 Inhibition Restricts HSV-1 IE Gene Transcription and Replication

doi: 10.3390/cells15050407

Figure Lengend Snippet: Backbone protein–protein interaction network under CDK1 inhibition during HSV-1 infection. Differentially phosphorylated proteins from the HSV-1/CDK1i vs. HSV-1/DMSO comparison were mapped to STRING (Homo sapiens; highest confidence, combined score ≥ 0.9) and imported into Cytoscape; node centralities were computed with cytoHubba. The backbone was defined as the union of nodes in the top decile for MCC, Degree, or Betweenness. CDK1 is shown at the center (red). Proteins annotated as transcription-related are arranged in the right ring (yellow); the remaining backbone proteins are grouped on the left into functional clusters based on curated Biological Processes. Nodes highlighted in green are known in vitro and in vivo CDK1 targets (motif-based). Edges depict STRING functional/physical interactions; line intensity reflects the STRING combined score; POLR2A is indicated with a black arrow. Programming language R (version 4.4.2) and RStudio integrated development environment (version 2024.12.0+467) were used to generate this image with select labels added using Microsoft PowerPoint 2016.

Article Snippet: POLR2A hypophosphorylation was monitored with primary antibodies specific to total POLR2A (Invitrogen, Carlsbad, CA, USA, cat. #MA1-10882; 1:1500 dilution) and phosphorylation-specific antibodies: phospho-Ser2-POLR2A (Novus Biologicals, Centennial, CO, USA, NBP2-59215; 1:1500 dilution) and phospho-Ser5-POLR2A (Abcam, Waltham, MA, USA, ab5408; 1500 dilution).

Techniques: Inhibition, Infection, Comparison, Functional Assay, In Vitro, In Vivo

Journal: Cells

Article Title: Phosphoproteome Remodeling upon CDK1 Inhibition Restricts HSV-1 IE Gene Transcription and Replication

doi: 10.3390/cells15050407

Figure Lengend Snippet: CDK1 inhibition decreases RNAPII CTD phosphorylation during early HSV-1 infection. ( a ) Schematic of RNAPII (POLR2A) CTD heptads 38–48 highlighting residues that were hypophosphorylated from our phosphoproteomic dataset (red boxes; approximate decreases shown in red, sites correspond to S2/T4/S4/S5 of the YSPTSPS consensus). ( b ) Immunoblot of POLR2A, Ser2-phosphorylated POLR2A (p-S2), and Ser5-phosphorylated POLR2A (p-S5) in infected cells treated with DMSO or CDK1i. β-actin served as a loading control; “−/+” indicates the absence/presence of CDK1i or HSV-1. Treated and untreated lanes shown are infected replicates. ( c ) Densitometry analyses of p-S2-POLR2A and p-S5-POLR2A; bands of the phosphorylated POLR2A were normalized to total POLR2A. For all panels, HFFs were pre-treated with CDK1i for 1 h, infected with HSV-1 (KOS, MOI = 2), and harvested at 3 hpi; M, PageRuler™ Plus Prestained Protein Ladder. Microsoft Excel 2016 and Microsoft PowerPoint 2016 were used to generate this figure.

Article Snippet: POLR2A hypophosphorylation was monitored with primary antibodies specific to total POLR2A (Invitrogen, Carlsbad, CA, USA, cat. #MA1-10882; 1:1500 dilution) and phosphorylation-specific antibodies: phospho-Ser2-POLR2A (Novus Biologicals, Centennial, CO, USA, NBP2-59215; 1:1500 dilution) and phospho-Ser5-POLR2A (Abcam, Waltham, MA, USA, ab5408; 1500 dilution).

Techniques: Inhibition, Phospho-proteomics, Infection, Western Blot, Control

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II transcription cycle in normal (N) tissue and tumor (T) of the large intestine. RNA expression level of Pol II transcription cycle-associated genes in GTEx and TCGA databases were analyzed. I initiation, E elongation, T termination. ( B ) Volcano plot for fold change of T vs N of 104 Pol II transcription-associated genes in COAD. NELFCD and SUPT4H1 genes are indicated with an arrow. NELFCD gene is highlighted in red. Statistical test: differential expression analysis was performed using DESeq2 (negative binomial generalized linear model with Wald test); P values were adjusted for multiple testing using the Benjamini–Hochberg method (adj.P.Val). The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . ( C ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is also indicated. Western blot image of NELF-C is reused in ( G ). ( D ) Western blot of SPT4-AID DLD-1 WCE using the indicated antibodies. The treatment time (h) of IAA is also indicated. ( E ) Western blot of NELF-C-AID DLD-1 cell nuclear fraction using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot images of NELF-C and Pol I are reused in Fig. . ( F ) Cell count ratios of 12, 24, and 36 h IAA to 0 h IAA are shown. Parental, NELF-C-AID, and SPT4-AID DLD-1 cells are displayed in light gray, red, and dark gray, respectively. Error bars represented the mean ± SEM (biological replicates, n = 4). Statistical test: paired two-sample t test (Student’s t test), two-sided, performed within each Group comparing the same Dish across adjacent timepoints (0 h vs 12 h, 12 h vs 24 h, 24 h vs 36 h) on the 0h-normalized ratios. P values are shown. n.s. (not significant, P > 0.05). ( G ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot image of NELF-C is reused in ( C ). ( H ) Cell cycle (%) of parental and NELF-C-AID DLD-1 cells following the indicated treatment time (h) of IAA. Error bars represented the mean ± SEM (biological replicates, n = 3). .

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression, Quantitative Proteomics, Western Blot, Cell Characterization

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Log 2 fold change of T vs N of the Pol II transcription-associated genes. Upregulated (>1) and down-regulated (<-1) genes are highlighted in red and blue, respectively. NELFCD gene is indicated with an arrow. ( B ) Volcano plot of the log2 fold change of Tumors (T) vs Normal tissues (N) on 835 cell cycle-related genes in COAD. CDKN1A , CDKN1B , and CDKN1C genes are indicated in red. The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. ( C ) Log2 of the RNA expression levels of NELFCD , SUPT4H1 , CDKN1A , and CDKN1C genes in N and T of the indicated tissues and tumors are compared. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Statistical test: Wilcoxon signed-rank test. P values are shown. The numbers of tumor (T) and normal (N) samples for each tissue types are provided in Table . ( D ) Log 2 fold change of T vs N of NELFCD across indicated tissue types. ( E ) Comparative proteomic analysis of NELF subunits and SPT4 in human primary colon cancers and its adjacent tissues. Statistical test: negative binomial distribution-based Wald test. P values are shown. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( F ) Schematic model of transcription addicted by NELF in tumor.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of POINT-seq strategy. Chromatin of NELF-C-AID or SPT4-AID DLD-1 cells was stringently isolated using Urea and Empigen detergent. Pol II intact nascent transcript (POINT) was precipitated with Pol II CTD antibody from DNA-digested chromatin fraction. ( B ) Example view of POINT-seq on RPS23 gene in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( C ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( D ) Violin plots of Termination Index (TI) in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median. ( E ) Example view of SIRV-normalized POINT-seq on RPS23 and HELLS genes in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( F ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( G ) Violin plots of TI in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. ** P = 0.0021, **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Isolation

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of TT-seq on PTPN13 gene in the indicated cell line treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. ( B ) Profiles of TT-seq mean signal in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. Data for 2 biological replicates with ≥300 kb genes are shown. ( C ) Box plots of elongation activity (EA) derived from TT-seq. Data for short and long genes are shown. n = 65. Statistical test: Wilcoxon rank-sum test. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). P value is 0.029. ( D ) Scatter plot showing the correlation between EA and TI. The regression line (red) and the 95% confidence interval (green) are also shown. n = 129. Statistical method: Spearman correlation. P value is 0.036. ( E ) Metagene of POINT-seq, TT-seq, Pol II EI (TT-seq/POINT-seq) for non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( F ) Box plots of Pol II EI (TT-seq/POINT-seq and TT-seq/PRO-seq) for non-overlapping pc genes (n = 3459) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Pol II EI for indicated three gene-length classes of normalized non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( H ) INTS3 ChIP-seq profile across normalized transcription units of non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( I ) Box plots of the log2 of the Dox/Ctrl termination index, two biological replicates in all genes, genes with transcription readthrough which is based on previous study (Dasilva et al, ), and NELF-C KD affected genes (top 25%, highlighted in orange box). 0: no change in TI index upon shCtrl or shINTS11 induction by Dox. The number of genes in each category is indicated on the figure. Statistical test: Kruskal–Wallis test. * P = 0.039, ** P = 0.0019, **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Activity Assay, Derivative Assay, ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II elongation index (EI) analysis. Pol II EI is determined as TT/POINT. 4sU (Green circle), Pol II (blue oval). ( B ) Pol II EI for all expressed pc genes across normalized transcription units in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( C ) Pol II EI for non-overlapping pc genes across LE. ( D ) Metagene of CSTF64 and XRN2 ChIP-seq for non-overlapping pc genes across normalized transcription units (top) and LE (bottom) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( E ) Quantification of CSTF64 and XRN2 ChIP-seq signals (IAA/DMSO) in 2.5 kb DoG regions of non-overlapping pc genes. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of SIRV-normalized POINT-seq on CD58 gene adjacent to a RI zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA replication initiation (RI) zone is highlighted in gray. RI score: 54.6579. Re-analyzed Pu-seq data shows RI zones in green. ( B ) Metagene analysis of POINT-seq on scaled RI zones −/+2.5 kb in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). ( C ) Box plots of SIRV-normalized POINT-seq signals in RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( D ) Scatter plots of SIRV-normalized POINT-seq signals and RI score in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). Cut-off value is Log 2 (1.25) in SIRV-normalized POINT-seq. Pol II-affected (P-A) and not affected (P-NA) zones are classified as higher and lower than the cut-off value, respectively. RI zones that are above the cut-off in the three timepoints (4, 12, 24 h IAA) are indicated in red. ( E ) Example view of SIRV-normalized POINT-seq on CD2AP gene adjacent to a P-NA zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA RI zone is highlighted in gray. RI score: 42.8401. ( F ) Box plots of SIRV-normalized POINT-seq signals in P-A ( n = 5878) and P-NA ( n = 6651) RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Size in bp of P-A ( n = 5878) or P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( H ) Distance in bp between the closest gene and the P-A ( n = 5878) and P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques:

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. Blue: Hoechst, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. The PLA was performed with indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. Statistical test: Wilcoxon rank-sum test. P values are shown. not significant (n.s.). ( C ) Western blot of chromatin fraction of NELF-C-AID DLD-1 cells (0, 4, and 24 h IAA) using the indicated antibodies. ( D ) Cell cycle (%) of 24 h DMSO or MLN-4924 treated NELF-C-AID DLD-1 cells. The cells were pre-treated with IAA or DMSO for 24 h. Error bars represented the mean ± SEM (biological replicates, n = 3). ( E ) BrdU dot blot assay to evaluate global DNA synthesis. ( F ) BrdU-IP-seq of two biological replicates (R1 and R2) for FOCAD gene regions in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. FOCAD DoG region is highlighted by gray. Early and Late S phase are separated by dashed line. ( G ) BrdU-IP-qPCR analysis to assess local DNA synthesis rates in region exhibiting termination defect. The top panel shows the locations of primer sets used for qPCR. The bottom panel displays the IP/input values for each locus, normalized to the ELAVL2 locus, a late-replicating region that is not expected to replicate at the onset of the S phase (ELAVL2 locus = 1). Error bars represented the mean ± SEM (biological replicates, n = 3). P values are indicated.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Western Blot, Dot Blot, DNA Synthesis

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in MCM2 (right) or PCNA (left) expressing NELF-C-AID DLD-1 cells. Blue: Hoechst, Green: MCM2 or PCNA, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in MCM2 or PCNA expressing NELF-C-AID DLD-1 cells. The PLA was performed with the indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. P values are shown. n.s. (not significant). ( C ) Schematic representation of the BrdU-IP assay performed following release from M phase. ( D ) BrdU-IP-seq of two biological replicates (R1 and R2) for the indicated chromosome 3 window in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Dashed lines indicate 1.25 in BrdU-IP (%). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. ( E ) Quantification of the POINT-seq (IAA 4 h – DMSO) signal over minus and plus BrdU zones located downstream of expressed pc genes (from TES + 2.5 to TES + 5 kb). Violin plots show the distribution (kernel density) of log10 POINT-seq read coverage mean values. Embedded box plots indicate the median (center; white dot/line) and the interquartile range (box; 25th–75th percentiles). Whiskers extend to 1.5×IQR; whisker endpoints represent the minimum and maximum values within this range. Statistical test: Brunner–Munzel test. **** P < 0.0001. ( F ) Model of NELF-mediated transcription termination and DNA replication initiation or elongation. With NELF, Pol II transcription is terminated at proximal PAS before Pol II reaches DNA RI zone. Without NELF, Pol II transcription is extended to distal PAS and impair replication in the RI zone. This perturbs DNA RI and/or causes T-R conflict (HO, Head-ON), resulting in cell quiescence.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Expressing, Whisker Assay