Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Rs79208225 SNP in HCC patients is associated with improved recurrence-free survival and decreased NELFE expression (A) Snapshot of Sanger sequencing from a wild type (WT) and a heterozygous NM_002904.6 :c.943-110C>T (rs79208225) variant carrying patient. (B) Kaplan-Meier 5 year recurrence-free survival curve for WT and rs79208225 SNP patients (WT n = 73, SNP n = 8, log rank test). (C) TaqMan RT-qPCR to evaluate NELFE mRNA expression in WT and rs79208225 SNP patient tumor (WT n = 32, SNP n = 8) and non-tumor (WT n = 33, SNP n = 6) tissue. Data are presented as mean ± SD. Student’s t test, ∗ p <0.05. (D) Representative images of hematoxylin & eosin and corresponding NELFE immunohistochemistry on WT or rs79208225 SNP patient tumors. Scale bars, 50 μm (WT n = 6, SNP n = 6). Data presented as mean ± SEM. p value from Welch’s test.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Expressing, DNA Sequencing, Variant Assay, Quantitative RT-PCR, Tissue, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Rs79208225 SNP promotes alternative splicing of NELFE exon 10 producing an uncharacterized NELFE mRNA isoform (A) Top, schematic of the minigene reporter. SNP minigenes have the rs79208225 C>T variant at the red line. WT minigenes retain the C allele. Arrows indicate primers for RT-PCR. Dotted line represents exon 10 skipping. Bottom, RT-PCR agarose gel of WT or rs79208225 SNP minigene transfected HCC cells. Rs79208225 SNP minigene cells contain a smaller band. CMV, cytomegalovirus; ex, exon; GFP, green fluorescent protein. (B) Top, schematic of the psiCheck dual luciferase firefly (fluc) and Renilla (Rluc) reporter for WT or rs79208225 SNP minigenes. Bottom, cells were transfected with WT or SNP reporter to quantify alternative splicing. Data presented as mean ± SD ( n = 3). Student’s t test, ∗ p <0.05; ∗∗ p <0.01. EV, empty vector control. (C) Digital PCR graph of d.ex10 mRNA isoform in HCC tumors from patients that harbor the rs79208225 SNP. Data presented as concentration (copies/μL) ± 95% confidence intervals. (D) RPIseq scores of the interaction probability of different splicing related proteins and the 41 nt sequence around the rs79208225 locus (20 nt on each flank) on the minus strand. Probability > 0.5 indicates positive interaction. SVM, support vector machine,; RF, random forest classifier. (E) Top, schematic of minigene reporters with RT-qPCR primers. Bottom, U2AF2 RNA immunoprecipitation PCR analyses of WT or SNP minigene transcripts. Data are presented as mean ± SD ( n = 3). Student’s t test, ∗∗ p <0.01 ; ∗∗∗ p <0.001. (F) RT-PCR agarose gel of FL NELFE and isoform at 48 h of MO treatment in HCC cell lines. (G) RT-qPCR detection of total NELFE mRNA and (H) d.ex10 isoform after 48 h of MO treatment. Total NELFE mRNA was evaluated with primers in exon 8. D.ex10 isoform was evaluated with a TaqMan probe for the exon9-exon11 junction. Data are presented as mean ± SD ( n = 4). Student’s t test, ∗∗ p <0.01, ∗∗∗ p <0.001. (I) Top, RT-PCR agarose gel of exon 10 deletion CRISPR model (mut) in Huh1 cells. Bottom, RT-qPCR analysis of total NELFE mRNA (left) and d.ex10 isoform (right) ( n = 4). Student’s t test, ∗∗ p <0.01; ∗∗∗∗ p <0.0001.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Alternative Splicing, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Luciferase, Plasmid Preparation, Control, Digital PCR, Concentration Assay, Sequencing, Quantitative RT-PCR, RNA Immunoprecipitation, CRISPR, Analysis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: d.ex10 mRNA isoform has a similar metabolism as FL mRNA transcripts and produces an uncharacterized NELFE protein isoform (A) Schematic of the effect of exon 10 skipping on NELFE stop codon location and 3′ UTR length. Depending on which poly(A) site is utilized, d.ex10 may have a 3′ UTR length of either 11 or 153 bp. Ex, exon; FL, full length; UTR, untranslated region. (B) mRNA stability evaluation of FL and d.ex10 mRNA transcripts following 5 μg/mL actinomycin D (Act. D) treatment in MO transfected cells or (C) 60 μM 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment in exon 10 CRISPR deletion model. Exon 10 included transcripts were detected with a TaqMan probe at the exon10-exon11 junction. D.ex10 transcripts were detected with a TaqMan probe at the exon9-exon11 junction. Dotted lines are at 50%. Data are presented as mean ± SD with one-phase decay curves ( n = 3 in B). (D) Top, schematics of the 3′ UTR psiCHECK-2 dual luciferase reporters. NELFE 3′ UTR sequences were cloned immediately downstream of Renilla’s (RLuc) stop codon. Bottom, HCC cells were transfected with dual luciferase constructs and relative luciferase expression was measured by normalizing Renilla luciferase signal to firefly luciferase signal. Data are presented as mean ± SEM ( n = 4). Student’s t test, ∗∗∗∗ p <0.0001. FLuc, firefly luciferase. (E) Representative immunoblots of NELF complex subunits after 72 h of MO treatment. (F) Representative immunoblots of NELF complex subunits in Huh1 WT and d.ex10 mutant (mut) cell lines. (G) Representative immunoblots of NELFE after 72 h of knockdown with either an siRNA targeting exon 4 (e4) or exon 10 (e10) to validate the lower band is a NELFE isoform that lacks exon 10 (d.ex10). (H) Left, representative immunoblot of FL or d.ex10 protein rescues tagged with GFP in stable shNELFE knockdown in Hep3B cells. Right, representative cell proliferation of Hep3B cells to assess effect of d.ex10 on cell phenotypes. Data are presented as mean ± SD ( n = 3). Student’s t test performed at 96 h. ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Stability, Transfection, CRISPR, Luciferase, Clone Assay, Construct, Expressing, Western Blot, Mutagenesis, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Loss of NELFE exon 10 is associated with reduced cancer cell phenotypes (A) Cell proliferation assay in MO treated HCC cell lines. Cells were plated for xCELLigence 48 h after MO transfection and measured every 12 for 72 h. Data are presented as mean ± SD ( n = 3–4). Statistical analysis was conducted at 72 h using a Student’s t test. ∗∗∗∗ p <0.0001. (B) Cell colony formation assay in MO treated HCC cell lines. Cells were plated in a 6-well plate 48 h after MO transfection. Left, quantification of colony formation after 10–14 days. Right, representative crystal violet stains of colonies. Data is presented as mean ± SD ( n = 3), Student’s t -test. ∗∗ p <0.01 and ∗∗∗∗ p <0.0001. (C) Spheroid formation assay in MO treated HCC cell lines. Cells were plated in a 6-well AlgiMatrix 48 h after MO transfection. Left, quantification of spheroid formation after 14 days. Right, representative spheroids imaged at 20×. Scale bars, 200 μm. Data are presented as mean ± SD ( n = 3). Statistical analysis was conducted by a Student’s t test. (D) Cell proliferation assay in Huh1 WT and Huh1 d.ex10 mut cell lines. Data are presented as mean ± SD ( n = 3). Student’s t test performed at 72 h. (E) Spheroid formation assay in Huh1 WT and Huh1 d.ex10 mut cell lines. Left, quantification of spheroid formation after 14 days. ∗∗∗∗ p <0.0001. Right, representative spheroids imaged at 20×. Scale bars, 200 μm. (F) MHCC97H tumor growth curve in athymic nude mice treated with intraperitoneal injections of 80 μM vivo-MOs. Gray arrows indicate treatment. Data presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 5). Two-way ANOVA with Dunnett’s multiple comparisons, ∗ p <0.05. (G) Graph representing tumor weights for indicated groups at the conclusion of the study. Data presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 5). Statistical analysis was conducted using a Student’s t test. (H) Left, RT-qPCR using a TaqMan probe spanning the exon 2-exon 3 junction to detect total NELFE mRNA from a less than 50 mg section of tumor tissue. Right, RT-qPCR using a TaqMan probe spanning the exon 9-exon 11 junction to detect d.ex10 mRNA transcripts. Data are presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 4). Student’s t test with Welch’s correction, ∗ p <0.05 (I) Representative hematoxylin and eosin (H&E) stain and NELFE immunohistochemistry of MHCC97H tumors with different vivo-MO treatments. Scale bars, 100 μm.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Proliferation Assay, Transfection, Analysis, Colony Assay, Tube Formation Assay, Spheroids, Quantitative RT-PCR, Tissue, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Targeted destruction of NELFE MO-loaded MBs extends survival in vivo (A) Schematic of locally released MOs from UTMD of MO loaded MBs. MO, morpholino; MB, microbubble; RO, retroorbital injection. (B) Microscopy images of Definity MBs loaded with fluoresceinated control vivo-MO. Images taken at 20×. Scale bars, 10 μm. (C) Ultrasound imaging of MB destruction and replenishment sequence. White circle outlines the tumor. Yellow line indicates the liver. (D) Immunoblot of Huh1 spheroids treated with two different concentrations of vivo-MO alone, loaded on MBs without UTMD, or loaded on MBs that subsequently underwent UTMD. NELFE band intensity was normalized to respective GAPDH then to WT. (E and F) Top, schematics of the experimental approach assessing MB + MO effect on Huh1.luc (E) and Huh7.luc (F) orthotopic tumors in NOD/SCID mice. Blue arrows indicate IVIS pre- and post-treatment imaging days. Black arrows indicate treatment days of UTMD of 50 μL MB + 80 μM MO administered through retro-orbital injections. Left, representative bioluminescence images of Huh1.luc (E) and Huh7.luc (F) mice pre-MB+MO treatment and 1-week post- final MB + MO treatment. Scale is in radiance (p/sec/cm/sr). Right, Kaplan-Meier survival curves of Huh1.luc (E) and Huh7.luc (F) vivo-MO treated mice (Huh1.luc experiment: MB + Ctrl n = 6, MB + i9 n = 8, MB + e10 n = 6; Huh7.luc experiment: MB + Ctrl n = 7, MB + i9 n = 5, MB + e10 n = 6). Survival analyses were conducted by a log-rank test.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: In Vivo, Injection, Microscopy, Control, Imaging, Sequencing, Western Blot, Spheroids

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: (A) Relative mRNA levels of YAP target genes (Cyr61, CTGF, ANKRD1) following siRNA-mediated knockdown of MePCE (7SKsnRNP subunit) in HEK293Ts (N=4). (B) Expression of Cyr61, CTGF, and ANKRD1 after HEXIM1/2 knockdown in HEK293Ts (N=4). (C) Expression of Cyr61, CTGF, and ANKRD1 following NELFA (NELF complex subunit) knockdown in HEK293Ts (N=4). (D) Expression of YAP target genes after NELFA knockdown in MDA-MB-231 cells (N=3). mRNA levels were quantified by RT-qPCR and normalised to GAPDH. Statistical analysis was performed using one-way ANOVA with multiple comparisons; p-values are indicated, with P<0.05 considered significant compared to control.

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

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

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) for MSigDB Oncogenic Signatures (C6) collection of significantly altered genes (p < 0.05) after NELFA knockdown compared to control knockdown. The Normalized Enrichment Score (NES) is plotted on the x-axis, with dot size corresponding to the number of genes overlapping with the ranked list, and color indicating statistical significance (−log₁₀ p -value), with red denoting higher significance. (B) GSEA Enrichment analysis for MSigDB HALLMARK Pathways. (C) Transcription Factor (TF) enrichment results from user-defined gene sets using a combination of perturbation and binding datasets. Y-axis lists the enrichment terms from Enrichr. Each term represents either a TF perturbation experiment (e.g., RNAi, overexpression) from public datasets (like GEO) or a TF binding profile from ENCODE ChIP-seq data (2015). Dot Size reflects the significance of enrichment with the z-score. Dot colour encodes the −log₁₀(p-value) from Fisher’s exact test, with red indicating higher significance. (D) Venn diagram showing the overlap of significantly differentially expressed genes (p < 0.05) across siNELFA, siYAP, and siNELFA-siYAP knockdowns compared to siControl. (E) Upset Plot showing the distribution of genes across four regulatory categories. Genes were classified based on log2FC≥ 1 thresholds from siNELFA, siYAP, and double knockdown comparisons. Each bar represents the number of genes exclusively assigned to one of the four categories. (F) Heatmap of genes grouped into four categories based on log2 fold change

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Knockdown, Control, Binding Assay, Over Expression, ChIP-sequencing

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: NELFA expression and its association with patient survival outcomes. (A) Representative immunohistochemistry (IHC) images of invasive ductal carcinoma (IDC) tumours displaying high (#505) and low (#376) NELFA expression, with scale bars of 500 µm (overview) and 100 µm (zoomed region) included for reference. (B-I) Kaplan-Meier (KM) plots are based on the NELFA expression cohort. Statistical significances are computed using the log-rank test (Mantel-Cox) in GraphPad Prism 8.0.1 (244). For each KM Plot, the number of patients in each NELFA expression category is listed, along with the number of events in parentheses. (B) Overall Survival of the IDC cohort, (C) Disease-free survival of the IDC cohort, (D-F) Overall Survival of the molecular subtypes, and (G-I) Disease-free survival of the molecular subtypes.

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet:

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Expressing

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: (A) Representative immunohistochemistry (IHC) images of invasive ductal carcinoma (IDC) tumours displaying high (#499) and low (#216) YAP expression, with scale bars of 500 µm (overview) and 100 µm (zoomed region) included for reference. (B-I) Kaplan-Meier (KM) plots are based on the YAP expression cohort. Statistical significances are computed using the log-rank test (Mantel-Cox) in GraphPad Prism 8.0.1 (244). For each KM Plot, the number of patients in each YAP expression category is listed, along with the number of events in parentheses. (B) Overall Survival of the IDC cohort, (C) Disease-free survival of the IDC cohort, (D-F) Overall Survival of the molecular subtypes, and (G-I) Disease-free survival of the molecular subtypes.

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: (A) Representative immunohistochemistry (IHC) images of invasive ductal carcinoma (IDC) tumours displaying combination of high and low YAP expression with high and low NELFA. YAP-High, NELFA-High (#499); YAP-High, NELFA-Low (#503); YAP-Low, NELFA-High (#92); YAP-Low, NELFA-Low (#288), with scale bars of 250 µm (overview) included for reference. (B-I) Kaplan-Meier (KM) plots are based on the combination of YAP and NELFA expression cohort. Statistical significances are computed using the log-rank test (Mantel-Cox) in GraphPad Prism 8.0.1 (244). For each KM Plot, the number of patients in each YAP and NELFA expression category is listed, along with the number of events in parentheses. (B) Overall Survival of the IDC cohort, (C) Disease-free survival of the IDC cohort, (D-F) Overall Survival of the molecular subtypes, and (G-I) Disease-free survival of the molecular subtypes.

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: NELFA-mediated promoter-proximal pausing restrains YAP-driven transcription and shapes context-dependent outcomes in breast cancer

doi: 10.64898/2025.12.29.696819

Figure Lengend Snippet: (A-C) KM plots of Breast cancer patients divided based expression of individual genes NELFA, YAP, and NELFA within High YAP expression of BC (Breast Cancer) IDC cohort. Statistical significances are computed using the log-rank test (Mantel-Cox) in GraphPad Prism 8.0.1 (244). (A) Overall and disease-free survival of NELFA expression for molecular subtypes of BC (B) Overall and disease-free survival of YAP expression for molecular subtypes of BC (C) Overall and disease-free survival of NELFA expression within high YAP for molecular subtypes of BC

Article Snippet: To optimize immunohistochemistry (IHC) conditions for NELFA (Santa Cruz, #sc-365004) and NELFB (Abcam, ab167401) antibodies, we tested epitope retrieval at pH levels of 6, 8, and 9 on high-tumor-content tissues using a 1:50 dilution of the antibody.

Techniques: Expressing