Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Bar graph showing the enriched pathways associated with the human proteins identified in the DENV NS3 samples. Metascape analysis was performed as in . b . Protein-protein interaction network showing the interaction between NS3 and human protein complexes involved in mRNA cleavage, polyadenylation and splicing processes. The presence of an edge denotes the detection of the human protein in the NS3 samples, and the thickness of the edge represents the average CRAPome score of the interaction from the replicate samples for NS3. c . Heat map depicting the average CRAPome scores of the CPSF and U5 spliceosome complex members detected in the DENV NS3 and NS5 samples. d . V5 immunoprecipitation of 293T cells transfected with the indicated FLAG-tagged DENV proteins and V5-tagged proteins involved in RNA metabolism, followed by Western blotting with the indicated antibodies showing the specific interaction between CPSF3, CPSF4 and EFTUD2, and DENV NS3 and NS5.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunoprecipitation, Transfection, Western Blot

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a. Immunofluorescence assay of Huh7 cells infected with DENV showing intracellular distribution of CPSF3 or CPSF4 (green) with infected cells indicated by staining with NS3 (red). b. Quantification of the fluorescent intensity for CPSF proteins (green), DENV NS3 (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism. c. Immunofluorescence assay of Huh7 cells expressing DENV NS3 showing the intracellular distribution of CPSF3 or CPSF4 (green) with cells expressing NS3 indicated by straining with NS3’s FLAG tag (red). d. Quantification of the fluorescent intensity for CPSF proteins (green), FLAG (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism. For b and d, thirty individual data points of the fluorescent intensity for the CPSF proteins were taken from the middle of the nucleus and cytoplasm, and compared by multiple unpaired t-tests and a p-value less than 0.05 was considered significant (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001). The images are representative of similar results from three independent biological experiments.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunofluorescence, Infection, Staining, Software, Expressing, FLAG-tag

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Immunofluorescence assay of DENV-infected Huh7 cells showing the intracellular distribution of CPSF4 (green) with infected cells indicated by staining with dsRNA (red). Quantification of the fluorescent intensity for CPSF proteins (green), dsRNA (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism (right). b. Immunofluorescence assay of Huh7 cells expressing DENV NS1 showing the levels of CPSF4 (green) in the nuclei of cells stained for the FLAG tag on NS1 (red). Quantification of the fluorescent intensity for CPSF proteins (green), FLAG (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism (right). Thirty individual data points of the fluorescent intensity for the CPSF proteins were taken from the middle of the nucleus and cytoplasm, and compared by multiple unpaired t-tests and a p-value less than 0.05 was considered significant (****, p < 0.0001). The images are representative of similar results from three independent biological experiments.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunofluorescence, Infection, Staining, Software, Expressing, FLAG-tag

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Bar graph (left) and dot plot (right) showing the relative levels of DENV genomic RNA and DENV plaque forming units (PFU) in Huh7 cells treated with the indicated siRNAs at 40μM for 24h before being infected with DENV for 24h. b. Bar graph (left) and dot plot (center) showing the relative levels of DENV genomic RNA and DENV plaque forming units (PFU) in Huh7 cells treated with increasing concentrations of siCPSF3-10. Bar graph showing the relative levels of CPSF3 mRNA and Western blot with the indicated antibodies showing the protein levels of DENV NS5 and CPSF3 (right). c . Bar graph (left) and dot plot (right) showing the relative levels of ZIKV genomic RNA and ZIKV plaque forming units (PFU) in Huh7 cells treated with the indicated siRNAs at 40μM for 24h before being infected with ZIKV for 24h. d . V5 immunoprecipitation of 293T cells transfected with FLAG-tagged ZIKV or DENV NS3 and V5-tagged CPSF3, followed by Western blotting with the indicated antibodies, showing that ZIKV NS3, like DENV NS3, interacts with CPSF3. e. Bar graph showing the relative cell viability of Huh7 cells treated with the indicated concentrations of JTE-607. f . Line graph showing the relative DENV infectivity of Huh7 cells treated with the indicated concentrations of JTE-607. The number of plaque forming units were counted for each sample and plotted relative to the untreated sample. The estimated EC 50 was calculated using Prism. Western blot with the indicated antibodies of the corresponding cell lysate samples showing the decrease in protein levels of DENV NS5, CPSF3, CPSF4 and actin with treatment of increasing concentrations of JTE-607. Mean values were compared to the respective control samples by Dunnett multiple comparison after one-way ANOVA, and a p-value less than 0.05 was considered significant (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Infection, Western Blot, Immunoprecipitation, Transfection, Control, Comparison

Journal: Journal of translational medicine

Article Title: CPSF4-mediated regulation of alternative splicing of HMG20B facilitates the progression of triple-negative breast cancer.

doi: 10.1186/s12967-024-06004-x

Figure Lengend Snippet: Fig. 2 CPSF4 Promotes Cell Proliferation and Migration in TNBC. A, B Cell proliferation of MDA-MB-231 and MDA-MB-468 cells transfected with siRNA-NC or si-CPSF4 detected by colony formation assay. C Cell proliferation of MDA-MB-231 cells transfected with overexpression negative control (OE-NC) or OE-CPSF4 detected by colony formation assay. D Transwell assay showing the migration of MDA-MB-231 cells with CPSF4 overexpression or knockdown. E Transwell assay showing the migration of MDA-MB-468 cells with CPSF4 overexpression or knockdown. **P < 0.01, ****P < 0.0001

Article Snippet: The primary antibodies used were CPSF4 antibody (15023-1-AP, Proteintech) and GAPDH antibody (60004-1-IG, Proteintech).

Techniques: Migration, Transfection, Colony Assay, Over Expression, Negative Control, Transwell Assay, Knockdown

Journal: Journal of translational medicine

Article Title: CPSF4-mediated regulation of alternative splicing of HMG20B facilitates the progression of triple-negative breast cancer.

doi: 10.1186/s12967-024-06004-x

Figure Lengend Snippet: Fig. 3 RNA sequencing (RNA-seq) analysis of transcriptome profiles regulated by CPSF4 in TNBC cells. A Evaluation of knockdown efficiency of si-CPSF4 in MDA-MB-231 cells by qRT-PCR. B The heatmap displays the hierarchical clustering of the Pearson correlation matrix for transcriptional expression levels in control and si-CPSF4 samples. C A volcano plot of differentially expressed genes associated with CPSF4, with upregulated and downregulated genes shown in orange and green, respectively. D Hierarchical clustering of differentially expressed genes (DEGs) in control and si-CPSF4 samples. E Top 10 kyoto encyclopedia of genes and genomes (KEGG) pathways enriched in upregulated genes. F Top 10 KEGG pathways enriched in downregulated genes. ****P < 0.0001

Article Snippet: The primary antibodies used were CPSF4 antibody (15023-1-AP, Proteintech) and GAPDH antibody (60004-1-IG, Proteintech).

Techniques: RNA Sequencing, Knockdown, Quantitative RT-PCR, Expressing, Control

Journal: Journal of translational medicine

Article Title: CPSF4-mediated regulation of alternative splicing of HMG20B facilitates the progression of triple-negative breast cancer.

doi: 10.1186/s12967-024-06004-x

Figure Lengend Snippet: Fig. 4 CPSF4 binding spectrum and binding motifs revealed by RNA immunoprecipitation sequencing (RIP-seq) analysis. A Western blot analysis of CPSF4 protein in MDA-MB-231 cells. B Distribution of reads across the entire reference genome. C Motif analysis using HOMER software showing the top 10 preferred binding motifs of CPSF4. **P < 0.01, ****P < 0.0001

Article Snippet: The primary antibodies used were CPSF4 antibody (15023-1-AP, Proteintech) and GAPDH antibody (60004-1-IG, Proteintech).

Techniques: Binding Assay, RNA Immunoprecipitation, Sequencing, Western Blot, Software

Journal: Journal of translational medicine

Article Title: CPSF4-mediated regulation of alternative splicing of HMG20B facilitates the progression of triple-negative breast cancer.

doi: 10.1186/s12967-024-06004-x

Figure Lengend Snippet: Fig. 6 CPSF4 Modulates Alternative Splicing Events (ASEs) in MDA-MB-231 cells. A Classification of all detected ASEs. B Classification of ASEs regulated by CPSF4. C Overlap between CPSF4-regulated DEGs and regulated alternative splicing genes (RASGs). D Top 10 biological processes associated with CPSF4-regulated alternative splicing genes as determined by GO enrichment analysis. E Top 10 pathways associated with CPSF4-regulated alternative splicing genes identified through KEGG analysis. F Venn diagram depicting the overlap between binding genes and RASGs

Article Snippet: The primary antibodies used were CPSF4 antibody (15023-1-AP, Proteintech) and GAPDH antibody (60004-1-IG, Proteintech).

Techniques: Alternative Splicing, Binding Assay