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: Molecular cell

Article Title: KAP1 is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs

doi: 10.1016/j.molcel.2020.04.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-NELFE , Santa Cruz Biotechnology , Cat# sc-377052; lot #L0117.

Techniques: Virus, Mutagenesis, Recombinant, Membrane, Protease Inhibitor, SYBR Green Assay, Western Blot, Staining, Plasmid Preparation, RNA HS Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, Cloning, shRNA, Software