Journal:

Article Title:

doi:

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:

Article Title:

doi:

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:

Article Title:

doi:

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:

Article Title:

doi:

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:

Article Title:

doi:

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