ilf3  (Proteintech)

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 expression is significantly upregulated and associated with poor prognosis in hepatocellular carcinoma. (A) ILF3 expression was analyzed utilizing GSE14429 and TCGA. (B) The relationship between the expression of ILF3 in HCC and tumor grade was analyzed using the UALCAN database based on TCGA. (C) Western blot assay was performed to detect the protein levels of ILF3 in HCC and adjacent normal tissues. (D) The protein expression levels of ILF3 detected via Western blot in HCC cell lines and immortalized human normal liver cells. (E) Relative ILF3 levels in HCC cell lines and immortalized human normal liver cells. (F) Immunohistochemistry was performed to detect ILF3 expression in HCC and adjacent normal tissues. (G) The association between ILF3 expression and overall survival and progression‐free survival was analyzed utilizing TCGA and GSE54236 . ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 promotes cell proliferation and EMT of hepatocellular carcinoma in vitro. (A) The expression levels of ILF3 were detected using Western blotting after transfection with lentivirus‐mediated shRNA. (B) CCK‐8 assays to assess cell proliferation after ILF3 knockdown in Hep3B and HepG2. (C) The colony formation assays were used to measure the colony‐forming ability of HCC cells after transfection with ILF3 knockdown shRNA. (D) The migration of Hep3B and HepG2 measured via wound healing assays after ILF3 knockdown. (E) The invasion of Hep3B and HepG2 measured via Transwell assays after ILF3 knockdown. (F) Western blot assays to evaluate E‐cadherin, N‐cadherin, and vimentin expression in Hep3B and HepG2 cells after ILF3 knockdown. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: In Vitro, Expressing, Western Blot, Transfection, shRNA, CCK-8 Assay, Knockdown, Migration

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 regulates in vivo growth and metastasis of hepatocellular carcinoma. (A) Tumors derived from xenograft mice were photographed. (B, C) The volume and weight of the tumor were measured after transfection with ILF3 knockdown shRNA. (D) The lung metastatic tumors derived from xenograft mice were photographed, and the number of lung metastatic nodules was recorded. (E) The survival time was monitored after injecting the ILF3‐knockdown HepG2 cells. (F) Western blot assay was performed to detect the expression of ILF3 in lung metastatic tissues derived from xenograft mice. (G) The expression of ILF3, E‐cadherin, N‐cadherin, and vimentin was measured in lung metastatic tissues using an immunohistochemistry assay. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: In Vivo, Derivative Assay, Transfection, Knockdown, shRNA, Western Blot, Expressing, Immunohistochemistry

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 interacts with HMGCL and promotes its degradation in a ubiquitin‐proteasome–dependent manner. (A) GSEA comparing the ILF3 low (blue) and high (red) expression subgroups of patients with HCC in TCGA indicated that the high expression of ILF3 showed a significant association with ubiquitin‐mediated proteolysis. (B) Co‐immunoprecipitation (Co‐IP) assay performed to show the interaction between ILF3 and HMGCL in Huh7, HepG2, and Hep3B cells. (C) Western blot assays showed that ILF3 decreased the expression of HMGCL in HCC cells. (D) RT‐qPCR showed that ILF3 had no effect on HMGCL mRNA levels. (E) The expression of HMGCL was measured in ILF3‐overexpressing cells and control cells after treatment with 50 μ g/mL cycloheximide. (F) The expression of HMGCL was measured in ILF3‐overexpressing cells and control cells via Western blot in Huh7 and Hep3B cells after treatment with 5 μ M MG132. (G) Co‐IP and Western blot were performed to measure the endogenous ILF3 ubiquitination in cells treated with 5 μ M MG132. ∗ p < 0.05, ∗∗ p < 0.01. Abbreviation: NS, not significant.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: Ubiquitin Proteomics, Expressing, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Control

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 promotes proliferation and invasion of hepatocellular carcinoma by inhibiting HMGCL expression. (A) Immunohistochemistry was performed to detect HMGCL expression in HCC and adjacent normal tissues. (B) Relative HMGCL levels in HCC cell lines and immortalized human normal liver cells. (C) CCK‐8 assay was performed to measure cell proliferation in Huh7 and HepG2 cells overexpressing ILF3 and control cells transfected with HMGCL‐overexpressing plasmids. (D) Transwell assay was performed to measure cell proliferation in Huh7 and HepG2 cells overexpressing ILF3 and control cells transfected with HMGCL‐overexpressing plasmids. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: Expressing, Immunohistochemistry, CCK-8 Assay, Control, Transfection, Transwell Assay

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: ILF3 promotes HMGCL ubiquitination by inhibiting the binding of the deubiquitinase USP38 to HMGCL. (A) Co‐immunoprecipitation (Co‐IP) and mass spectrometry analyses were performed to identify proteins interacting with both ILF3 and HMGCL. Gene enrichment analysis was performed to identify proteins belonging to the “ubiquitin‐protein ligase” category. (B) Score Sequest HT and gene enrichment of the candidates. (C) Co‐IP assay performed to show the interaction between HMGCL and USP38 in Huh7 and HepG2 cells. (D) Huh7 and HepG2 cells were transfected with USP38 wild‐type or USP38‐CAHA (inactive mutant) plasmids, and Co‐IP and Western blotting were performed to measure endogenous HMGCL ubiquitination after treatment with 5 μ M MG132. (E) Huh7 and HepG2 cells were transfected with siRNA or ILF3‐overexpressing plasmids. Western blot assay was performed to measure the effect of ILF3 on USP38 and HMGCL. Additional Co‐IP assay was performed to measure the protein binding capacity between USP38 and HMGCL.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: Ubiquitin Proteomics, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Mass Spectrometry, Transfection, Mutagenesis, Western Blot, Protein Binding

Journal: Human Mutation

Article Title: ILF3 Regulates Cell Proliferation and Metastasis by Competitively Antagonizing the Interaction Between HMGCL and USP38 in Hepatocellular Carcinoma

doi: 10.1155/humu/2654435

Figure Lengend Snippet: Working model of ILF3 facilitating the proliferation and metastasis of HCC. Deubiquitinase USP38 interacts with HMGCL and contributes to HMGCL deubiquitination. ILF3 is elevated in HCC and facilitates the interaction with HMGCL. ILF3 competitively antagonizes the interaction between USP38 and HMGCL, thereby reducing USP38‐mediated HMGCL deubiquitination and decreasing HMGCL protein abundance. Finally, ILF3 promotes cell proliferation and metastasis in HCC.

Article Snippet: The ILF3 knockdown and overexpression recombinant lentiviruses were purchased from Genechem (Shanghai, China) to construct stable transfection cell lines.

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Results from genome-wide loss-of-function screens in human pluripotent stem cells (hPSCs) during pluripotency exit induced by either TGFβ and bFGF withdrawal or MAPK pathway inhibition , depicting mean Z-score from three replicates. ( b ) CRISPRi (top) and representative western blot with signal quantification (bottom) depicting validation of ILF2 and ILF3 knockdown efficiency after 3 days of doxycycline treatment in hPSCs expressing targeted guide RNAs. ( c ) Experimental design for pluripotency exit assays. ( d ) Representative flow cytometry quantification of NANOG in hPSCs under self-renewal and exit conditions by MAPK pathway inhibition following ILF2 or ILF3 knockdown. ( e ) Schematic of chimpanzee ( Pan troglodytes ) PSC differentiation experiments. ( f ) Representative flow cytometry quantification of NANOG-positive chimpanzee PSCs under self-renewal or exit conditions following ILF2 or ILF3 knockdown. ( g ) Schematic of mouse ESC differentiation experiments. ( h ) Representative flow cytometry quantification of NANOG-positive mouse ESCs under primed pluripotency or exit conditions following Ilf2 or Ilf3 knockdown. ( i ) Hierarchical clustering of RNA-seq datasets from hPSCs in self-renewal and exit conditions. ( j ) Gene Set Enrichment Analysis (GSEA) of pluripotency-associated genes in ILF2 -or ILF3 -depleted cells versus control during exit conditions (ILF2: NES=2.50, p=3.58E-13; ILF3: NES=2.66, p= 1.54E-13). ( k ) Differential gene expression analysis comparing control and ILF2 or ILF3 -knockdown hPSCs after exit from pluripotency (n = 2 biological replicates; fold change > 1.5; P < 0.05). Red and blue indicate up-and down-regulated genes, respectively. ( l ) Quantification of pluripotency-associated differentially accessible regions in control and ILF2 or ILF3 -depleted cells under self-renewal and exit conditions (n = 2 biological replicates, P values calculated using paired Wilcoxon rank-sum test). ( m ) Experimental design for three-dimensional human peri-gastruloid formation. ( n ) Representative brightfield images of ILF2 -and ILF3 -depleted human peri-gastruloids. Scale bar, 100 µm. ( o ) Analysis of longest axis length ILF2 -and ILF3 -depleted human peri-gastruloids (n = 7-10 biological replicates; mean ± s.d; P values determined by two-way ANOVA with Šidák’s multiple comparisons test). ( p ) Immunofluorescence analysis of human peri-gastruloids showing SOX2 (red), SOX17 (green), T (pink), and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted cells. Scale bar, 50 μm.

Article Snippet: Co-immunoprecipitation (Co-IP) of ILF3 and ILF2 was performed as previously described (see above) using 6 μg rabbit anti-ILF3 antibody (Bethyl Laboratories, A303-651A), 6 μg rabbit anti-NF45 antibody (Bethyl Laboratories, A303-147A), or 6 μg rabbit IgG (Sigma-Aldrich, 12-370).

Techniques: Genome Wide, Inhibition, Western Blot, Biomarker Discovery, Knockdown, Expressing, Flow Cytometry, RNA Sequencing, Control, Gene Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of neuronal differentiation showing TUJ1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted neural progenitor cells (NPCs). Scale bar, 100 µm. ( b ) Quantification of TUJ1-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( c ) Gene Ontology enrichment analysis of Biological Processes (BP) of downregulated genes in ILF2 -and ILF3 -depleted neurons compared to controls (two-tailed Fisher’s exact test). ( d ) Differential gene expression analysis in neurons following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( e ) Immunofluorescence analysis of endodermal differentiation showing SOX17 expression (red) and nuclear DAPI staining (blue) in control and ILF2 / 3 -depleted foregut progenitors. Scale bar, 100 µm. ( f ) Quantification of SOX17-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( g ) GSEA of endoderm-specific genes in ILF2 -or ILF3 -depleted foregut cells (shILF2: NES =-1.94, P = 2.44E-4; shILF3: NES =-1.93, P = 1.44E-4). ( h ) Differential gene expression analysis in foregut cells following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( i ) Immunofluorescence analysis of myogenic differentiation showing MYH1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted primary myoblasts. Scale bar, 100 µm. ( j ) Quantification of MYH1-positive cells (n = 8 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( k ) GSEA of myoblast differentiation genes in ILF2 -or ILF3 -depleted myotubes (shILF2: NES =-1.57, P = 0.0224; shILF3: NES =-1.68, P = 5.86E-3). ( l ) Differential gene expression analysis in myotubes following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction) Red and blue indicate up-and down-regulated genes, respectively

Article Snippet: Co-immunoprecipitation (Co-IP) of ILF3 and ILF2 was performed as previously described (see above) using 6 μg rabbit anti-ILF3 antibody (Bethyl Laboratories, A303-651A), 6 μg rabbit anti-NF45 antibody (Bethyl Laboratories, A303-147A), or 6 μg rabbit IgG (Sigma-Aldrich, 12-370).

Techniques: Immunofluorescence, Expressing, Staining, Control, Two Tailed Test, Gene Expression, Knockdown, Cell Characterization

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Genome-wide distribution of ILF3 binding sites determined by CUT&Tag analysis, showing normalized read density across gene bodies ±3 kb from transcriptional centers. ( b ) Overlap between ILF3 binding sites identified by CUT&Tag and eCLIP analyses (RPKM>0.5; top 1% of peaks by area under the curve (AUC). ( c ) Gene Ontology enrichment analysis of ILF3-bound regions identified by eCLIP-seq and CUT&Tag. (d) Distribution of ILF3 eCLIP signal intensity relative to size-matched input controls (log₂FC > 3; P < 0.001). ( e ) Enrichment analysis of repetitive element classes in ILF3 eCLIP peaks relative to a randomized peak distribution. ( f ) AlphaFold3-predicted structural model of the ILF2/3 complex bound to Alu RNA. ( g ) Proteomic analysis of ILF3 interactors identified by immunoprecipitation-mass spectrometry (n = 3 biological replicates). ( h ) Single nucleotide variants (SNVs) detected in ILF3-depleted versus control hPSCs. Base substitution types include counts for the reverse complement variant. ( i ) Comparison of editing frequencies at A-to-I edited sites in ILF3 knockdown or control knockdown hPSCs. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( j ) Aggregate plot showing ILF2/3 eCLIP signal distribution centered around A-to-I-edited sites after ILF3 depletion. Z-scores calculated relative to a randomized peak distribution. ( k ) Phylogenetic tree – the diameter of each bubble is proportional to the percentage of each species’ respective genome that aligns to Alu elements (top row) or ILF3 eCLIP targets identified in hPSCs (bottom row). ( l ) Western blot analysis of interactions between FLAG-tagged ADAR1 and wild-type HA-ILF3 or RNA-binding mutant (HA-ILF3ΔRBM). ( m ) Editing frequencies in ILF3 wild-type versus ΔRBM rescue conditions (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( n ) Quantification of NANOG-positive cells under self-renewal and exit conditions following rescue with wild-type or ΔRBM mutant ILF3 (n = 3 biological replicates; P values determined by one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Co-immunoprecipitation (Co-IP) of ILF3 and ILF2 was performed as previously described (see above) using 6 μg rabbit anti-ILF3 antibody (Bethyl Laboratories, A303-651A), 6 μg rabbit anti-NF45 antibody (Bethyl Laboratories, A303-147A), or 6 μg rabbit IgG (Sigma-Aldrich, 12-370).

Techniques: Genome Wide, Binding Assay, Immunoprecipitation, Mass Spectrometry, Control, Variant Assay, Comparison, Knockdown, Western Blot, RNA Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Generation of ILF3-FKBP12 F36V hPSCs using CRISPR-Cas9-mediated knock-in (top) and western blot showing ILF3 and ILF2 protein levels after 24 hours of dTAG V -1 treatment (bottom). ( b ) Western blot analysis of fractionated whole cell and subcellular lysates. ( c ) Editing frequencies in chromatin-associated mRNAs after 24 hours of ILF3 degradation (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( d ) Aggregate plot showing ILF3 eCLIP signal distribution centered around A-to-I-edited sites in chromatin-associated mRNAs after 24 hours of ILF3 degradation. Z-scores calculated relative to a randomized peak distribution. ( e ) Alternative splicing events in chromatin-associated mRNAs 24 hours after ILF3 degradation (|ΔPSI| > 0.1, FDR < 0.05). ( f ) Enrichment of repetitive elements in cassette exons more included in chromatin-associated mRNAs after ILF3 degradation (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( g ) Editing frequencies in transcripts showing increased exon inclusion after ILF3 degradation in chromatin-associated mRNAs (ΔPSI > 0.1, FDR < 0.05; n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( h ) Experimental design for splicing analysis in CRISPRi ILF3 hPSCs after rescue with ILF3 WT or ILF3ΔRBM. ( i ) Alternative splicing comparison between ILF3ΔRBM and wild-type rescue in ILF2 / 3 -depleted cells (|ΔPSI| > 0.1, FDR < 0.05). ( j ) Enrichment of repetitive elements in cassette exons more included in ILF3ΔRBM versus wild-type rescue (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( k ) Experimental design for editing and splicing analysis after ILF3 degradation and ADAR knockdown. ( l ) Editing frequencies at A-to-I sites observed after 24 hours of ILF3 degradation in ADAR knockdown hPSCs, relative to events observed in control knockdown cells. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( m ) Alternative splicing ratios 24 hours after ILF3 degradation in ADAR knockdown hPSCs, relative to inclusion events observed in control knockdown cells (ΔPSI > 0.1, FDR < 0.05; P values determined by Wilcoxon rank-sum test). ( n ) Representative flow cytometric analysis of NANOG-positive cells under self-renewal and exit conditions following ILF3 silencing and rescue with control or ADAR knockdown.

Article Snippet: Co-immunoprecipitation (Co-IP) of ILF3 and ILF2 was performed as previously described (see above) using 6 μg rabbit anti-ILF3 antibody (Bethyl Laboratories, A303-651A), 6 μg rabbit anti-NF45 antibody (Bethyl Laboratories, A303-147A), or 6 μg rabbit IgG (Sigma-Aldrich, 12-370).

Techniques: CRISPR, Knock-In, Western Blot, Alternative Splicing, Comparison, Knockdown, Control

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) RNA-seq expression analysis of mis-spliced transcripts after 24 (left) and 96 hours (right) of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; |FC| > 1.5, P < 0.05). ( b ) IsoformSwitch analysis of PTC-containing vs. non-PTC-containing transcript isoforms after 24 hours of ILF3 degradation, divided by ILF3 eCLIP targets (left) and non-targets (right). P values determined by Kolmogorov-Smirnoff test. ( c ) Experimental design for transcript analysis after ILF3 degradation and UPF1 knockdown. ( d ) RT-qPCR analysis of mis-spliced transcripts with and without UPF1 knockdown (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Tukey’s multiple comparisons test). ( e ) Proteomic analysis of mis-spliced transcripts after 24 and 96 hours of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; P < 0.05). ( f ) GO analysis of downregulated proteins associated with mis-spliced transcripts. ( g ) RT-qPCR analysis of NANOG expression after knockdown of chromatin regulators in hPSCs under exit conditions induced by MAPK pathway inhibition (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Dunnett’s multiple comparisons test). ( h ) RT-qPCR analysis of pluripotency gene expression after control or ILF3 knockdown with and without rescue by indicated genes in hPSCs under exit conditions (n = 3 biological replicates). ( i ) Experimental design for chromatin and histone mark analysis after ILF3 degradation. ( j ) ATAC-seq analysis after 96 hours of ILF3 degradation (n = 2 biological replicates; red: increased accessibility, blue: decreased accessibility; |FC| > 1.5, P < 0.05). ( k ) HOMER motif analysis at differential chromatin accessibility regions (n = 2 biological replicates; |FC| > 1.5; P < 0.05). ( l ) Aggregate histone modification profiles after 96 hours of ILF3 degradation (n = 3 biological replicates) versus control. ( m ) Representative CUT&Tag tracks showing changes in histone mark deposition at pluripotency (left) and differentiation (right) genes. ( n , o ) Proposed mechanistic model (AS = Alternative spliced).

Article Snippet: Co-immunoprecipitation (Co-IP) of ILF3 and ILF2 was performed as previously described (see above) using 6 μg rabbit anti-ILF3 antibody (Bethyl Laboratories, A303-651A), 6 μg rabbit anti-NF45 antibody (Bethyl Laboratories, A303-147A), or 6 μg rabbit IgG (Sigma-Aldrich, 12-370).

Techniques: RNA Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Inhibition, Gene Expression, Control, Modification

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Results from genome-wide loss-of-function screens in human pluripotent stem cells (hPSCs) during pluripotency exit induced by either TGFβ and bFGF withdrawal or MAPK pathway inhibition , depicting mean Z-score from three replicates. ( b ) CRISPRi (top) and representative western blot with signal quantification (bottom) depicting validation of ILF2 and ILF3 knockdown efficiency after 3 days of doxycycline treatment in hPSCs expressing targeted guide RNAs. ( c ) Experimental design for pluripotency exit assays. ( d ) Representative flow cytometry quantification of NANOG in hPSCs under self-renewal and exit conditions by MAPK pathway inhibition following ILF2 or ILF3 knockdown. ( e ) Schematic of chimpanzee ( Pan troglodytes ) PSC differentiation experiments. ( f ) Representative flow cytometry quantification of NANOG-positive chimpanzee PSCs under self-renewal or exit conditions following ILF2 or ILF3 knockdown. ( g ) Schematic of mouse ESC differentiation experiments. ( h ) Representative flow cytometry quantification of NANOG-positive mouse ESCs under primed pluripotency or exit conditions following Ilf2 or Ilf3 knockdown. ( i ) Hierarchical clustering of RNA-seq datasets from hPSCs in self-renewal and exit conditions. ( j ) Gene Set Enrichment Analysis (GSEA) of pluripotency-associated genes in ILF2 -or ILF3 -depleted cells versus control during exit conditions (ILF2: NES=2.50, p=3.58E-13; ILF3: NES=2.66, p= 1.54E-13). ( k ) Differential gene expression analysis comparing control and ILF2 or ILF3 -knockdown hPSCs after exit from pluripotency (n = 2 biological replicates; fold change > 1.5; P < 0.05). Red and blue indicate up-and down-regulated genes, respectively. ( l ) Quantification of pluripotency-associated differentially accessible regions in control and ILF2 or ILF3 -depleted cells under self-renewal and exit conditions (n = 2 biological replicates, P values calculated using paired Wilcoxon rank-sum test). ( m ) Experimental design for three-dimensional human peri-gastruloid formation. ( n ) Representative brightfield images of ILF2 -and ILF3 -depleted human peri-gastruloids. Scale bar, 100 µm. ( o ) Analysis of longest axis length ILF2 -and ILF3 -depleted human peri-gastruloids (n = 7-10 biological replicates; mean ± s.d; P values determined by two-way ANOVA with Šidák’s multiple comparisons test). ( p ) Immunofluorescence analysis of human peri-gastruloids showing SOX2 (red), SOX17 (green), T (pink), and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted cells. Scale bar, 50 μm.

Article Snippet: Lysates were subjected to standard Western blotting procedures using the following primary antibodies: rabbit anti-human/mouse ILF2 (1:3000, Bethyl A303-147A), rabbit anti-human/mouse ILF3 (1:3000, Bethyl A303-651), anti-FLAG (1:2000, Addgene 194502), anti-HA (1:2000, BioLegend 901516), HRP-rabbit anti-human/mouse β-actin (1:3000, Cell Signaling 5125), and rabbit anti-human/mouse vinculin (1:2000, Cell Signaling 13901S).

Techniques: Genome Wide, Inhibition, Western Blot, Biomarker Discovery, Knockdown, Expressing, Flow Cytometry, RNA Sequencing, Control, Gene Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of neuronal differentiation showing TUJ1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted neural progenitor cells (NPCs). Scale bar, 100 µm. ( b ) Quantification of TUJ1-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( c ) Gene Ontology enrichment analysis of Biological Processes (BP) of downregulated genes in ILF2 -and ILF3 -depleted neurons compared to controls (two-tailed Fisher’s exact test). ( d ) Differential gene expression analysis in neurons following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( e ) Immunofluorescence analysis of endodermal differentiation showing SOX17 expression (red) and nuclear DAPI staining (blue) in control and ILF2 / 3 -depleted foregut progenitors. Scale bar, 100 µm. ( f ) Quantification of SOX17-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( g ) GSEA of endoderm-specific genes in ILF2 -or ILF3 -depleted foregut cells (shILF2: NES =-1.94, P = 2.44E-4; shILF3: NES =-1.93, P = 1.44E-4). ( h ) Differential gene expression analysis in foregut cells following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( i ) Immunofluorescence analysis of myogenic differentiation showing MYH1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted primary myoblasts. Scale bar, 100 µm. ( j ) Quantification of MYH1-positive cells (n = 8 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( k ) GSEA of myoblast differentiation genes in ILF2 -or ILF3 -depleted myotubes (shILF2: NES =-1.57, P = 0.0224; shILF3: NES =-1.68, P = 5.86E-3). ( l ) Differential gene expression analysis in myotubes following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction) Red and blue indicate up-and down-regulated genes, respectively

Article Snippet: Lysates were subjected to standard Western blotting procedures using the following primary antibodies: rabbit anti-human/mouse ILF2 (1:3000, Bethyl A303-147A), rabbit anti-human/mouse ILF3 (1:3000, Bethyl A303-651), anti-FLAG (1:2000, Addgene 194502), anti-HA (1:2000, BioLegend 901516), HRP-rabbit anti-human/mouse β-actin (1:3000, Cell Signaling 5125), and rabbit anti-human/mouse vinculin (1:2000, Cell Signaling 13901S).

Techniques: Immunofluorescence, Expressing, Staining, Control, Two Tailed Test, Gene Expression, Knockdown, Cell Characterization

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Genome-wide distribution of ILF3 binding sites determined by CUT&Tag analysis, showing normalized read density across gene bodies ±3 kb from transcriptional centers. ( b ) Overlap between ILF3 binding sites identified by CUT&Tag and eCLIP analyses (RPKM>0.5; top 1% of peaks by area under the curve (AUC). ( c ) Gene Ontology enrichment analysis of ILF3-bound regions identified by eCLIP-seq and CUT&Tag. (d) Distribution of ILF3 eCLIP signal intensity relative to size-matched input controls (log₂FC > 3; P < 0.001). ( e ) Enrichment analysis of repetitive element classes in ILF3 eCLIP peaks relative to a randomized peak distribution. ( f ) AlphaFold3-predicted structural model of the ILF2/3 complex bound to Alu RNA. ( g ) Proteomic analysis of ILF3 interactors identified by immunoprecipitation-mass spectrometry (n = 3 biological replicates). ( h ) Single nucleotide variants (SNVs) detected in ILF3-depleted versus control hPSCs. Base substitution types include counts for the reverse complement variant. ( i ) Comparison of editing frequencies at A-to-I edited sites in ILF3 knockdown or control knockdown hPSCs. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( j ) Aggregate plot showing ILF2/3 eCLIP signal distribution centered around A-to-I-edited sites after ILF3 depletion. Z-scores calculated relative to a randomized peak distribution. ( k ) Phylogenetic tree – the diameter of each bubble is proportional to the percentage of each species’ respective genome that aligns to Alu elements (top row) or ILF3 eCLIP targets identified in hPSCs (bottom row). ( l ) Western blot analysis of interactions between FLAG-tagged ADAR1 and wild-type HA-ILF3 or RNA-binding mutant (HA-ILF3ΔRBM). ( m ) Editing frequencies in ILF3 wild-type versus ΔRBM rescue conditions (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( n ) Quantification of NANOG-positive cells under self-renewal and exit conditions following rescue with wild-type or ΔRBM mutant ILF3 (n = 3 biological replicates; P values determined by one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Lysates were subjected to standard Western blotting procedures using the following primary antibodies: rabbit anti-human/mouse ILF2 (1:3000, Bethyl A303-147A), rabbit anti-human/mouse ILF3 (1:3000, Bethyl A303-651), anti-FLAG (1:2000, Addgene 194502), anti-HA (1:2000, BioLegend 901516), HRP-rabbit anti-human/mouse β-actin (1:3000, Cell Signaling 5125), and rabbit anti-human/mouse vinculin (1:2000, Cell Signaling 13901S).

Techniques: Genome Wide, Binding Assay, Immunoprecipitation, Mass Spectrometry, Control, Variant Assay, Comparison, Knockdown, Western Blot, RNA Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Generation of ILF3-FKBP12 F36V hPSCs using CRISPR-Cas9-mediated knock-in (top) and western blot showing ILF3 and ILF2 protein levels after 24 hours of dTAG V -1 treatment (bottom). ( b ) Western blot analysis of fractionated whole cell and subcellular lysates. ( c ) Editing frequencies in chromatin-associated mRNAs after 24 hours of ILF3 degradation (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( d ) Aggregate plot showing ILF3 eCLIP signal distribution centered around A-to-I-edited sites in chromatin-associated mRNAs after 24 hours of ILF3 degradation. Z-scores calculated relative to a randomized peak distribution. ( e ) Alternative splicing events in chromatin-associated mRNAs 24 hours after ILF3 degradation (|ΔPSI| > 0.1, FDR < 0.05). ( f ) Enrichment of repetitive elements in cassette exons more included in chromatin-associated mRNAs after ILF3 degradation (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( g ) Editing frequencies in transcripts showing increased exon inclusion after ILF3 degradation in chromatin-associated mRNAs (ΔPSI > 0.1, FDR < 0.05; n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( h ) Experimental design for splicing analysis in CRISPRi ILF3 hPSCs after rescue with ILF3 WT or ILF3ΔRBM. ( i ) Alternative splicing comparison between ILF3ΔRBM and wild-type rescue in ILF2 / 3 -depleted cells (|ΔPSI| > 0.1, FDR < 0.05). ( j ) Enrichment of repetitive elements in cassette exons more included in ILF3ΔRBM versus wild-type rescue (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( k ) Experimental design for editing and splicing analysis after ILF3 degradation and ADAR knockdown. ( l ) Editing frequencies at A-to-I sites observed after 24 hours of ILF3 degradation in ADAR knockdown hPSCs, relative to events observed in control knockdown cells. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( m ) Alternative splicing ratios 24 hours after ILF3 degradation in ADAR knockdown hPSCs, relative to inclusion events observed in control knockdown cells (ΔPSI > 0.1, FDR < 0.05; P values determined by Wilcoxon rank-sum test). ( n ) Representative flow cytometric analysis of NANOG-positive cells under self-renewal and exit conditions following ILF3 silencing and rescue with control or ADAR knockdown.

Article Snippet: Lysates were subjected to standard Western blotting procedures using the following primary antibodies: rabbit anti-human/mouse ILF2 (1:3000, Bethyl A303-147A), rabbit anti-human/mouse ILF3 (1:3000, Bethyl A303-651), anti-FLAG (1:2000, Addgene 194502), anti-HA (1:2000, BioLegend 901516), HRP-rabbit anti-human/mouse β-actin (1:3000, Cell Signaling 5125), and rabbit anti-human/mouse vinculin (1:2000, Cell Signaling 13901S).

Techniques: CRISPR, Knock-In, Western Blot, Alternative Splicing, Comparison, Knockdown, Control

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) RNA-seq expression analysis of mis-spliced transcripts after 24 (left) and 96 hours (right) of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; |FC| > 1.5, P < 0.05). ( b ) IsoformSwitch analysis of PTC-containing vs. non-PTC-containing transcript isoforms after 24 hours of ILF3 degradation, divided by ILF3 eCLIP targets (left) and non-targets (right). P values determined by Kolmogorov-Smirnoff test. ( c ) Experimental design for transcript analysis after ILF3 degradation and UPF1 knockdown. ( d ) RT-qPCR analysis of mis-spliced transcripts with and without UPF1 knockdown (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Tukey’s multiple comparisons test). ( e ) Proteomic analysis of mis-spliced transcripts after 24 and 96 hours of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; P < 0.05). ( f ) GO analysis of downregulated proteins associated with mis-spliced transcripts. ( g ) RT-qPCR analysis of NANOG expression after knockdown of chromatin regulators in hPSCs under exit conditions induced by MAPK pathway inhibition (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Dunnett’s multiple comparisons test). ( h ) RT-qPCR analysis of pluripotency gene expression after control or ILF3 knockdown with and without rescue by indicated genes in hPSCs under exit conditions (n = 3 biological replicates). ( i ) Experimental design for chromatin and histone mark analysis after ILF3 degradation. ( j ) ATAC-seq analysis after 96 hours of ILF3 degradation (n = 2 biological replicates; red: increased accessibility, blue: decreased accessibility; |FC| > 1.5, P < 0.05). ( k ) HOMER motif analysis at differential chromatin accessibility regions (n = 2 biological replicates; |FC| > 1.5; P < 0.05). ( l ) Aggregate histone modification profiles after 96 hours of ILF3 degradation (n = 3 biological replicates) versus control. ( m ) Representative CUT&Tag tracks showing changes in histone mark deposition at pluripotency (left) and differentiation (right) genes. ( n , o ) Proposed mechanistic model (AS = Alternative spliced).

Article Snippet: Lysates were subjected to standard Western blotting procedures using the following primary antibodies: rabbit anti-human/mouse ILF2 (1:3000, Bethyl A303-147A), rabbit anti-human/mouse ILF3 (1:3000, Bethyl A303-651), anti-FLAG (1:2000, Addgene 194502), anti-HA (1:2000, BioLegend 901516), HRP-rabbit anti-human/mouse β-actin (1:3000, Cell Signaling 5125), and rabbit anti-human/mouse vinculin (1:2000, Cell Signaling 13901S).

Techniques: RNA Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Inhibition, Gene Expression, Control, Modification

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Results from genome-wide loss-of-function screens in human pluripotent stem cells (hPSCs) during pluripotency exit induced by either TGFβ and bFGF withdrawal or MAPK pathway inhibition , depicting mean Z-score from three replicates. ( b ) CRISPRi (top) and representative western blot with signal quantification (bottom) depicting validation of ILF2 and ILF3 knockdown efficiency after 3 days of doxycycline treatment in hPSCs expressing targeted guide RNAs. ( c ) Experimental design for pluripotency exit assays. ( d ) Representative flow cytometry quantification of NANOG in hPSCs under self-renewal and exit conditions by MAPK pathway inhibition following ILF2 or ILF3 knockdown. ( e ) Schematic of chimpanzee ( Pan troglodytes ) PSC differentiation experiments. ( f ) Representative flow cytometry quantification of NANOG-positive chimpanzee PSCs under self-renewal or exit conditions following ILF2 or ILF3 knockdown. ( g ) Schematic of mouse ESC differentiation experiments. ( h ) Representative flow cytometry quantification of NANOG-positive mouse ESCs under primed pluripotency or exit conditions following Ilf2 or Ilf3 knockdown. ( i ) Hierarchical clustering of RNA-seq datasets from hPSCs in self-renewal and exit conditions. ( j ) Gene Set Enrichment Analysis (GSEA) of pluripotency-associated genes in ILF2 -or ILF3 -depleted cells versus control during exit conditions (ILF2: NES=2.50, p=3.58E-13; ILF3: NES=2.66, p= 1.54E-13). ( k ) Differential gene expression analysis comparing control and ILF2 or ILF3 -knockdown hPSCs after exit from pluripotency (n = 2 biological replicates; fold change > 1.5; P < 0.05). Red and blue indicate up-and down-regulated genes, respectively. ( l ) Quantification of pluripotency-associated differentially accessible regions in control and ILF2 or ILF3 -depleted cells under self-renewal and exit conditions (n = 2 biological replicates, P values calculated using paired Wilcoxon rank-sum test). ( m ) Experimental design for three-dimensional human peri-gastruloid formation. ( n ) Representative brightfield images of ILF2 -and ILF3 -depleted human peri-gastruloids. Scale bar, 100 µm. ( o ) Analysis of longest axis length ILF2 -and ILF3 -depleted human peri-gastruloids (n = 7-10 biological replicates; mean ± s.d; P values determined by two-way ANOVA with Šidák’s multiple comparisons test). ( p ) Immunofluorescence analysis of human peri-gastruloids showing SOX2 (red), SOX17 (green), T (pink), and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted cells. Scale bar, 50 μm.

Article Snippet: For immunoprecipitation, fresh Protein G Mag Sepharose beads were blocked overnight in 1% BSA (Sigma-Aldrich) at 4°C, incubated with 6 μg ILF3 (Bethyl A303-651A) antibody or 6 μg rabbit IgG (Sigma-Aldrich, 12-370) for 2 hours at room temperature, and crosslinked using 20 mM DMP in 0.2 M triethanolamine for 30 minutes at room temperature.

Techniques: Genome Wide, Inhibition, Western Blot, Biomarker Discovery, Knockdown, Expressing, Flow Cytometry, RNA Sequencing, Control, Gene Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of neuronal differentiation showing TUJ1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted neural progenitor cells (NPCs). Scale bar, 100 µm. ( b ) Quantification of TUJ1-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( c ) Gene Ontology enrichment analysis of Biological Processes (BP) of downregulated genes in ILF2 -and ILF3 -depleted neurons compared to controls (two-tailed Fisher’s exact test). ( d ) Differential gene expression analysis in neurons following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( e ) Immunofluorescence analysis of endodermal differentiation showing SOX17 expression (red) and nuclear DAPI staining (blue) in control and ILF2 / 3 -depleted foregut progenitors. Scale bar, 100 µm. ( f ) Quantification of SOX17-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( g ) GSEA of endoderm-specific genes in ILF2 -or ILF3 -depleted foregut cells (shILF2: NES =-1.94, P = 2.44E-4; shILF3: NES =-1.93, P = 1.44E-4). ( h ) Differential gene expression analysis in foregut cells following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( i ) Immunofluorescence analysis of myogenic differentiation showing MYH1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted primary myoblasts. Scale bar, 100 µm. ( j ) Quantification of MYH1-positive cells (n = 8 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( k ) GSEA of myoblast differentiation genes in ILF2 -or ILF3 -depleted myotubes (shILF2: NES =-1.57, P = 0.0224; shILF3: NES =-1.68, P = 5.86E-3). ( l ) Differential gene expression analysis in myotubes following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction) Red and blue indicate up-and down-regulated genes, respectively

Article Snippet: For immunoprecipitation, fresh Protein G Mag Sepharose beads were blocked overnight in 1% BSA (Sigma-Aldrich) at 4°C, incubated with 6 μg ILF3 (Bethyl A303-651A) antibody or 6 μg rabbit IgG (Sigma-Aldrich, 12-370) for 2 hours at room temperature, and crosslinked using 20 mM DMP in 0.2 M triethanolamine for 30 minutes at room temperature.

Techniques: Immunofluorescence, Expressing, Staining, Control, Two Tailed Test, Gene Expression, Knockdown, Cell Characterization

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Genome-wide distribution of ILF3 binding sites determined by CUT&Tag analysis, showing normalized read density across gene bodies ±3 kb from transcriptional centers. ( b ) Overlap between ILF3 binding sites identified by CUT&Tag and eCLIP analyses (RPKM>0.5; top 1% of peaks by area under the curve (AUC). ( c ) Gene Ontology enrichment analysis of ILF3-bound regions identified by eCLIP-seq and CUT&Tag. (d) Distribution of ILF3 eCLIP signal intensity relative to size-matched input controls (log₂FC > 3; P < 0.001). ( e ) Enrichment analysis of repetitive element classes in ILF3 eCLIP peaks relative to a randomized peak distribution. ( f ) AlphaFold3-predicted structural model of the ILF2/3 complex bound to Alu RNA. ( g ) Proteomic analysis of ILF3 interactors identified by immunoprecipitation-mass spectrometry (n = 3 biological replicates). ( h ) Single nucleotide variants (SNVs) detected in ILF3-depleted versus control hPSCs. Base substitution types include counts for the reverse complement variant. ( i ) Comparison of editing frequencies at A-to-I edited sites in ILF3 knockdown or control knockdown hPSCs. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( j ) Aggregate plot showing ILF2/3 eCLIP signal distribution centered around A-to-I-edited sites after ILF3 depletion. Z-scores calculated relative to a randomized peak distribution. ( k ) Phylogenetic tree – the diameter of each bubble is proportional to the percentage of each species’ respective genome that aligns to Alu elements (top row) or ILF3 eCLIP targets identified in hPSCs (bottom row). ( l ) Western blot analysis of interactions between FLAG-tagged ADAR1 and wild-type HA-ILF3 or RNA-binding mutant (HA-ILF3ΔRBM). ( m ) Editing frequencies in ILF3 wild-type versus ΔRBM rescue conditions (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( n ) Quantification of NANOG-positive cells under self-renewal and exit conditions following rescue with wild-type or ΔRBM mutant ILF3 (n = 3 biological replicates; P values determined by one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: For immunoprecipitation, fresh Protein G Mag Sepharose beads were blocked overnight in 1% BSA (Sigma-Aldrich) at 4°C, incubated with 6 μg ILF3 (Bethyl A303-651A) antibody or 6 μg rabbit IgG (Sigma-Aldrich, 12-370) for 2 hours at room temperature, and crosslinked using 20 mM DMP in 0.2 M triethanolamine for 30 minutes at room temperature.

Techniques: Genome Wide, Binding Assay, Immunoprecipitation, Mass Spectrometry, Control, Variant Assay, Comparison, Knockdown, Western Blot, RNA Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Generation of ILF3-FKBP12 F36V hPSCs using CRISPR-Cas9-mediated knock-in (top) and western blot showing ILF3 and ILF2 protein levels after 24 hours of dTAG V -1 treatment (bottom). ( b ) Western blot analysis of fractionated whole cell and subcellular lysates. ( c ) Editing frequencies in chromatin-associated mRNAs after 24 hours of ILF3 degradation (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( d ) Aggregate plot showing ILF3 eCLIP signal distribution centered around A-to-I-edited sites in chromatin-associated mRNAs after 24 hours of ILF3 degradation. Z-scores calculated relative to a randomized peak distribution. ( e ) Alternative splicing events in chromatin-associated mRNAs 24 hours after ILF3 degradation (|ΔPSI| > 0.1, FDR < 0.05). ( f ) Enrichment of repetitive elements in cassette exons more included in chromatin-associated mRNAs after ILF3 degradation (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( g ) Editing frequencies in transcripts showing increased exon inclusion after ILF3 degradation in chromatin-associated mRNAs (ΔPSI > 0.1, FDR < 0.05; n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( h ) Experimental design for splicing analysis in CRISPRi ILF3 hPSCs after rescue with ILF3 WT or ILF3ΔRBM. ( i ) Alternative splicing comparison between ILF3ΔRBM and wild-type rescue in ILF2 / 3 -depleted cells (|ΔPSI| > 0.1, FDR < 0.05). ( j ) Enrichment of repetitive elements in cassette exons more included in ILF3ΔRBM versus wild-type rescue (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( k ) Experimental design for editing and splicing analysis after ILF3 degradation and ADAR knockdown. ( l ) Editing frequencies at A-to-I sites observed after 24 hours of ILF3 degradation in ADAR knockdown hPSCs, relative to events observed in control knockdown cells. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( m ) Alternative splicing ratios 24 hours after ILF3 degradation in ADAR knockdown hPSCs, relative to inclusion events observed in control knockdown cells (ΔPSI > 0.1, FDR < 0.05; P values determined by Wilcoxon rank-sum test). ( n ) Representative flow cytometric analysis of NANOG-positive cells under self-renewal and exit conditions following ILF3 silencing and rescue with control or ADAR knockdown.

Article Snippet: For immunoprecipitation, fresh Protein G Mag Sepharose beads were blocked overnight in 1% BSA (Sigma-Aldrich) at 4°C, incubated with 6 μg ILF3 (Bethyl A303-651A) antibody or 6 μg rabbit IgG (Sigma-Aldrich, 12-370) for 2 hours at room temperature, and crosslinked using 20 mM DMP in 0.2 M triethanolamine for 30 minutes at room temperature.

Techniques: CRISPR, Knock-In, Western Blot, Alternative Splicing, Comparison, Knockdown, Control

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) RNA-seq expression analysis of mis-spliced transcripts after 24 (left) and 96 hours (right) of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; |FC| > 1.5, P < 0.05). ( b ) IsoformSwitch analysis of PTC-containing vs. non-PTC-containing transcript isoforms after 24 hours of ILF3 degradation, divided by ILF3 eCLIP targets (left) and non-targets (right). P values determined by Kolmogorov-Smirnoff test. ( c ) Experimental design for transcript analysis after ILF3 degradation and UPF1 knockdown. ( d ) RT-qPCR analysis of mis-spliced transcripts with and without UPF1 knockdown (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Tukey’s multiple comparisons test). ( e ) Proteomic analysis of mis-spliced transcripts after 24 and 96 hours of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; P < 0.05). ( f ) GO analysis of downregulated proteins associated with mis-spliced transcripts. ( g ) RT-qPCR analysis of NANOG expression after knockdown of chromatin regulators in hPSCs under exit conditions induced by MAPK pathway inhibition (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Dunnett’s multiple comparisons test). ( h ) RT-qPCR analysis of pluripotency gene expression after control or ILF3 knockdown with and without rescue by indicated genes in hPSCs under exit conditions (n = 3 biological replicates). ( i ) Experimental design for chromatin and histone mark analysis after ILF3 degradation. ( j ) ATAC-seq analysis after 96 hours of ILF3 degradation (n = 2 biological replicates; red: increased accessibility, blue: decreased accessibility; |FC| > 1.5, P < 0.05). ( k ) HOMER motif analysis at differential chromatin accessibility regions (n = 2 biological replicates; |FC| > 1.5; P < 0.05). ( l ) Aggregate histone modification profiles after 96 hours of ILF3 degradation (n = 3 biological replicates) versus control. ( m ) Representative CUT&Tag tracks showing changes in histone mark deposition at pluripotency (left) and differentiation (right) genes. ( n , o ) Proposed mechanistic model (AS = Alternative spliced).

Article Snippet: For immunoprecipitation, fresh Protein G Mag Sepharose beads were blocked overnight in 1% BSA (Sigma-Aldrich) at 4°C, incubated with 6 μg ILF3 (Bethyl A303-651A) antibody or 6 μg rabbit IgG (Sigma-Aldrich, 12-370) for 2 hours at room temperature, and crosslinked using 20 mM DMP in 0.2 M triethanolamine for 30 minutes at room temperature.

Techniques: RNA Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Inhibition, Gene Expression, Control, Modification

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Results from genome-wide loss-of-function screens in human pluripotent stem cells (hPSCs) during pluripotency exit induced by either TGFβ and bFGF withdrawal or MAPK pathway inhibition , depicting mean Z-score from three replicates. ( b ) CRISPRi (top) and representative western blot with signal quantification (bottom) depicting validation of ILF2 and ILF3 knockdown efficiency after 3 days of doxycycline treatment in hPSCs expressing targeted guide RNAs. ( c ) Experimental design for pluripotency exit assays. ( d ) Representative flow cytometry quantification of NANOG in hPSCs under self-renewal and exit conditions by MAPK pathway inhibition following ILF2 or ILF3 knockdown. ( e ) Schematic of chimpanzee ( Pan troglodytes ) PSC differentiation experiments. ( f ) Representative flow cytometry quantification of NANOG-positive chimpanzee PSCs under self-renewal or exit conditions following ILF2 or ILF3 knockdown. ( g ) Schematic of mouse ESC differentiation experiments. ( h ) Representative flow cytometry quantification of NANOG-positive mouse ESCs under primed pluripotency or exit conditions following Ilf2 or Ilf3 knockdown. ( i ) Hierarchical clustering of RNA-seq datasets from hPSCs in self-renewal and exit conditions. ( j ) Gene Set Enrichment Analysis (GSEA) of pluripotency-associated genes in ILF2 -or ILF3 -depleted cells versus control during exit conditions (ILF2: NES=2.50, p=3.58E-13; ILF3: NES=2.66, p= 1.54E-13). ( k ) Differential gene expression analysis comparing control and ILF2 or ILF3 -knockdown hPSCs after exit from pluripotency (n = 2 biological replicates; fold change > 1.5; P < 0.05). Red and blue indicate up-and down-regulated genes, respectively. ( l ) Quantification of pluripotency-associated differentially accessible regions in control and ILF2 or ILF3 -depleted cells under self-renewal and exit conditions (n = 2 biological replicates, P values calculated using paired Wilcoxon rank-sum test). ( m ) Experimental design for three-dimensional human peri-gastruloid formation. ( n ) Representative brightfield images of ILF2 -and ILF3 -depleted human peri-gastruloids. Scale bar, 100 µm. ( o ) Analysis of longest axis length ILF2 -and ILF3 -depleted human peri-gastruloids (n = 7-10 biological replicates; mean ± s.d; P values determined by two-way ANOVA with Šidák’s multiple comparisons test). ( p ) Immunofluorescence analysis of human peri-gastruloids showing SOX2 (red), SOX17 (green), T (pink), and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted cells. Scale bar, 50 μm.

Article Snippet: The whole cell lysates were sonicated and subjected to limited digestion with RNase I (40 U/mL of lysate), followed by immunoprecipitation for ILF2 or ILF3-RNA complexes using anti-ILF2 (10 μg Bethyl Laboratories A303-147A) or anti-ILF3 antibodies (10 μg Bethyl Laboratories A303-651A).

Techniques: Genome Wide, Inhibition, Western Blot, Biomarker Discovery, Knockdown, Expressing, Flow Cytometry, RNA Sequencing, Control, Gene Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of neuronal differentiation showing TUJ1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted neural progenitor cells (NPCs). Scale bar, 100 µm. ( b ) Quantification of TUJ1-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( c ) Gene Ontology enrichment analysis of Biological Processes (BP) of downregulated genes in ILF2 -and ILF3 -depleted neurons compared to controls (two-tailed Fisher’s exact test). ( d ) Differential gene expression analysis in neurons following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( e ) Immunofluorescence analysis of endodermal differentiation showing SOX17 expression (red) and nuclear DAPI staining (blue) in control and ILF2 / 3 -depleted foregut progenitors. Scale bar, 100 µm. ( f ) Quantification of SOX17-positive cells (n = 10 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( g ) GSEA of endoderm-specific genes in ILF2 -or ILF3 -depleted foregut cells (shILF2: NES =-1.94, P = 2.44E-4; shILF3: NES =-1.93, P = 1.44E-4). ( h ) Differential gene expression analysis in foregut cells following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction). Red and blue indicate up-and down-regulated genes, respectively. ( i ) Immunofluorescence analysis of myogenic differentiation showing MYH1 expression (green) and nuclear DAPI staining (blue) in control and ILF2 or ILF3 -depleted primary myoblasts. Scale bar, 100 µm. ( j ) Quantification of MYH1-positive cells (n = 8 independent images per condition; P values determined by unpaired two-tailed Student’s t-test). ( k ) GSEA of myoblast differentiation genes in ILF2 -or ILF3 -depleted myotubes (shILF2: NES =-1.57, P = 0.0224; shILF3: NES =-1.68, P = 5.86E-3). ( l ) Differential gene expression analysis in myotubes following ILF2 or ILF3 knockdown (n = 2 biological replicates; |fold change| > 1.5; P < 0.05, Wald test with Benjamini-Hochberg correction) Red and blue indicate up-and down-regulated genes, respectively

Article Snippet: The whole cell lysates were sonicated and subjected to limited digestion with RNase I (40 U/mL of lysate), followed by immunoprecipitation for ILF2 or ILF3-RNA complexes using anti-ILF2 (10 μg Bethyl Laboratories A303-147A) or anti-ILF3 antibodies (10 μg Bethyl Laboratories A303-651A).

Techniques: Immunofluorescence, Expressing, Staining, Control, Two Tailed Test, Gene Expression, Knockdown, Cell Characterization

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Genome-wide distribution of ILF3 binding sites determined by CUT&Tag analysis, showing normalized read density across gene bodies ±3 kb from transcriptional centers. ( b ) Overlap between ILF3 binding sites identified by CUT&Tag and eCLIP analyses (RPKM>0.5; top 1% of peaks by area under the curve (AUC). ( c ) Gene Ontology enrichment analysis of ILF3-bound regions identified by eCLIP-seq and CUT&Tag. (d) Distribution of ILF3 eCLIP signal intensity relative to size-matched input controls (log₂FC > 3; P < 0.001). ( e ) Enrichment analysis of repetitive element classes in ILF3 eCLIP peaks relative to a randomized peak distribution. ( f ) AlphaFold3-predicted structural model of the ILF2/3 complex bound to Alu RNA. ( g ) Proteomic analysis of ILF3 interactors identified by immunoprecipitation-mass spectrometry (n = 3 biological replicates). ( h ) Single nucleotide variants (SNVs) detected in ILF3-depleted versus control hPSCs. Base substitution types include counts for the reverse complement variant. ( i ) Comparison of editing frequencies at A-to-I edited sites in ILF3 knockdown or control knockdown hPSCs. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( j ) Aggregate plot showing ILF2/3 eCLIP signal distribution centered around A-to-I-edited sites after ILF3 depletion. Z-scores calculated relative to a randomized peak distribution. ( k ) Phylogenetic tree – the diameter of each bubble is proportional to the percentage of each species’ respective genome that aligns to Alu elements (top row) or ILF3 eCLIP targets identified in hPSCs (bottom row). ( l ) Western blot analysis of interactions between FLAG-tagged ADAR1 and wild-type HA-ILF3 or RNA-binding mutant (HA-ILF3ΔRBM). ( m ) Editing frequencies in ILF3 wild-type versus ΔRBM rescue conditions (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( n ) Quantification of NANOG-positive cells under self-renewal and exit conditions following rescue with wild-type or ΔRBM mutant ILF3 (n = 3 biological replicates; P values determined by one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: The whole cell lysates were sonicated and subjected to limited digestion with RNase I (40 U/mL of lysate), followed by immunoprecipitation for ILF2 or ILF3-RNA complexes using anti-ILF2 (10 μg Bethyl Laboratories A303-147A) or anti-ILF3 antibodies (10 μg Bethyl Laboratories A303-651A).

Techniques: Genome Wide, Binding Assay, Immunoprecipitation, Mass Spectrometry, Control, Variant Assay, Comparison, Knockdown, Western Blot, RNA Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) Generation of ILF3-FKBP12 F36V hPSCs using CRISPR-Cas9-mediated knock-in (top) and western blot showing ILF3 and ILF2 protein levels after 24 hours of dTAG V -1 treatment (bottom). ( b ) Western blot analysis of fractionated whole cell and subcellular lysates. ( c ) Editing frequencies in chromatin-associated mRNAs after 24 hours of ILF3 degradation (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( d ) Aggregate plot showing ILF3 eCLIP signal distribution centered around A-to-I-edited sites in chromatin-associated mRNAs after 24 hours of ILF3 degradation. Z-scores calculated relative to a randomized peak distribution. ( e ) Alternative splicing events in chromatin-associated mRNAs 24 hours after ILF3 degradation (|ΔPSI| > 0.1, FDR < 0.05). ( f ) Enrichment of repetitive elements in cassette exons more included in chromatin-associated mRNAs after ILF3 degradation (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( g ) Editing frequencies in transcripts showing increased exon inclusion after ILF3 degradation in chromatin-associated mRNAs (ΔPSI > 0.1, FDR < 0.05; n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( h ) Experimental design for splicing analysis in CRISPRi ILF3 hPSCs after rescue with ILF3 WT or ILF3ΔRBM. ( i ) Alternative splicing comparison between ILF3ΔRBM and wild-type rescue in ILF2 / 3 -depleted cells (|ΔPSI| > 0.1, FDR < 0.05). ( j ) Enrichment of repetitive elements in cassette exons more included in ILF3ΔRBM versus wild-type rescue (ΔPSI > 0.1, FDR < 0.05) relative to a randomized peak distribution. ( k ) Experimental design for editing and splicing analysis after ILF3 degradation and ADAR knockdown. ( l ) Editing frequencies at A-to-I sites observed after 24 hours of ILF3 degradation in ADAR knockdown hPSCs, relative to events observed in control knockdown cells. (n = 2 biological replicates; P values determined by Wilcoxon rank-sum test). ( m ) Alternative splicing ratios 24 hours after ILF3 degradation in ADAR knockdown hPSCs, relative to inclusion events observed in control knockdown cells (ΔPSI > 0.1, FDR < 0.05; P values determined by Wilcoxon rank-sum test). ( n ) Representative flow cytometric analysis of NANOG-positive cells under self-renewal and exit conditions following ILF3 silencing and rescue with control or ADAR knockdown.

Article Snippet: The whole cell lysates were sonicated and subjected to limited digestion with RNase I (40 U/mL of lysate), followed by immunoprecipitation for ILF2 or ILF3-RNA complexes using anti-ILF2 (10 μg Bethyl Laboratories A303-147A) or anti-ILF3 antibodies (10 μg Bethyl Laboratories A303-651A).

Techniques: CRISPR, Knock-In, Western Blot, Alternative Splicing, Comparison, Knockdown, Control

Journal: bioRxiv

Article Title: Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

doi: 10.64898/2026.01.14.699349

Figure Lengend Snippet: ( a ) RNA-seq expression analysis of mis-spliced transcripts after 24 (left) and 96 hours (right) of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; |FC| > 1.5, P < 0.05). ( b ) IsoformSwitch analysis of PTC-containing vs. non-PTC-containing transcript isoforms after 24 hours of ILF3 degradation, divided by ILF3 eCLIP targets (left) and non-targets (right). P values determined by Kolmogorov-Smirnoff test. ( c ) Experimental design for transcript analysis after ILF3 degradation and UPF1 knockdown. ( d ) RT-qPCR analysis of mis-spliced transcripts with and without UPF1 knockdown (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Tukey’s multiple comparisons test). ( e ) Proteomic analysis of mis-spliced transcripts after 24 and 96 hours of ILF3 degradation (FDR < 0.05; ΔPSI > 0.1; P < 0.05). ( f ) GO analysis of downregulated proteins associated with mis-spliced transcripts. ( g ) RT-qPCR analysis of NANOG expression after knockdown of chromatin regulators in hPSCs under exit conditions induced by MAPK pathway inhibition (n = 3 biological replicates; mean ± s.d.; P values determined by one-way ANOVA with Dunnett’s multiple comparisons test). ( h ) RT-qPCR analysis of pluripotency gene expression after control or ILF3 knockdown with and without rescue by indicated genes in hPSCs under exit conditions (n = 3 biological replicates). ( i ) Experimental design for chromatin and histone mark analysis after ILF3 degradation. ( j ) ATAC-seq analysis after 96 hours of ILF3 degradation (n = 2 biological replicates; red: increased accessibility, blue: decreased accessibility; |FC| > 1.5, P < 0.05). ( k ) HOMER motif analysis at differential chromatin accessibility regions (n = 2 biological replicates; |FC| > 1.5; P < 0.05). ( l ) Aggregate histone modification profiles after 96 hours of ILF3 degradation (n = 3 biological replicates) versus control. ( m ) Representative CUT&Tag tracks showing changes in histone mark deposition at pluripotency (left) and differentiation (right) genes. ( n , o ) Proposed mechanistic model (AS = Alternative spliced).

Article Snippet: The whole cell lysates were sonicated and subjected to limited digestion with RNase I (40 U/mL of lysate), followed by immunoprecipitation for ILF2 or ILF3-RNA complexes using anti-ILF2 (10 μg Bethyl Laboratories A303-147A) or anti-ILF3 antibodies (10 μg Bethyl Laboratories A303-651A).

Techniques: RNA Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Inhibition, Gene Expression, Control, Modification