Journal: Cell reports
Article Title: Nucleocytoplasmic transport of the RNA-binding protein CELF2 regulates neural stem cell fates.
doi: 10.1016/j.celrep.2021.109226
Figure Lengend Snippet: Figure 1. CELF2 pathogenic variants disrupt nucleocytoplasmic localization and cortical development (A) Selected MR images. Sagittal T1-weighted (top row) and coronal T2-weighted (bottom row) images of patient 1 (i and i0), patient 2 (ii and ii0), patient 3 (iii and iii0), and patient 4 (iv and iv0) are shown. The images were obtained at age 25 months for patient 1, 16 months for patient 2, 25 months for patient 3, and 41 months for patient 4. All patients have simplified gyral pattern, which is mild in patients 1, 3, and 4, and is moderate in patient 2. The corpus callosum is healthy in patient 1, normally developed but diffusely thinned in patient 2, healthy in patient 3, and mildly hypoplastic in patient 4. Patients 1 and 3 have normally developed anterior commissure (arrows in i, ii0, iii, and iii0), but patients 2 and 4 have an absence of the anterior commissure. Patient 2 has moderate ventriculomegaly. (B) The schematic showing the location of the identified variants in the coding exons and the corresponding parts of the CELF2 protein with three RNA-recognition motifs (RRM1–3) (Ladd and Cooper, 2004). The affected residues are within a known nuclear-localization signal (NLS) in RRM3. (C) Boxplots showing CELFs expression in human cortical NPCs (PAX6+ and SOX2+) (single-cell RNA-seq dataset from Pollen et al., 2015). (D) Temporal quantitative real-time PCR analysis of the expression of CELF1 and CELF2 in the developing mouse cortices at the indicated time points. n = 3 samples at each time point. (E) Confocal images of HEK293 cells expressing EGFP-CELF2 (green) that carry different pathogenic variants and counterstained for Hoechst 33258 (blue). ‘‘N’’ denotes the nucleus. Dashed white lines denote nuclei (blue). The intensity of CELF2 immunosignals across individual cells is shown at the bottom with the nucleus region labeled by the blue bar. Scale bar, 10 mm. (F) RT-PCR analysis of exon-skipping events for PPP1R mRNA and TRAF3 mRNA in HEK293 cells transfected with empty vector (control), wild-type (WT), or mutant CELF2. (G) Quantifications of the percentage of transcripts with the skipped exon. One-way ANOVA with Tukey’s test. n = 3 experiments. (H) The quantitative real-time PCR enrichment analysis of GAPDH mRNA and MCL mRNA by RIP with control IgG or an anti-EGFP antibody in HEK293 cells transfected with EGFP-tagged CELF2-NES or mutant CELF2 (normalized to the total RNA input). n = 3 experiments. Data are presented as means ± SEM. Each symbol represents one experiment.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Sox2 (goat) R&D Systems AF2018 bIII-tubulin (mouse) Biolegend 801201 CELF2 (rabbit) Abcam ab186430 CELF2 (mouse) Santa Cruz Biotechnology sc47731 Ki67 (mouse) BD Biosciences 550609 CSDE1 (rabbit) Abcam ab201688 LaminB1 (mouse) Abcam ab8982 Lap2 (mouse) BD Biosciences 611000 Satb2 (mouse) Abcam ab51502 Tbr2 (rabbit) Abcam ab183991 Tbr2 (rat) Invitrogen 14-4875-80 Pax6 (rabbit) Biolegend 901301 pH3 (mouse) Invitrogen MA5-15220 pH3 (mouse) Cell Signaling 9706s GFP (mouse) Abcam ab290 GFP (chicken) EMD Millipore AB16901 GAPDH (rabbit) Sigma G9545 Dcp1a (mouse) Abnova H00055802-M06 4E-T Abnova H00056478-B01 Bacterial strains pEGFP-C1 Clontech Discontinued pCAGIG Matsuda and Cepko, 2004 Addgene (11159) pSUPER OligoEngine VEC-pBS-0002 Chemicals, peptides, and recombinant proteins Q5 Site-Directed Mutagenesis Kit NEB E0554S Polyethylenimine Sigma Aldrich 765090 FGF2 Corning CB-40060A B27 Thermo Fisher 17504044 Penicillin-streptomycin Wisent 450-201-EL L-glutamine Wisent 609-065-EL DMEM Thermo Fisher 11995065 Neurobasal Thermo Fisher 21103049 FBS Wisent 098-150 Lipofectamine Stem Thermo Fisher STEM00015 Laminin Corning 354232 Poly-D-Lysine Sigma Aldrich P6407 Trizol Invitrogen LS15596026 Maxima H Minus cDNA Synthesis Kit Thermo Fisher M1681 Phusion High Fidelity PCR Master Mix Thermo Fisher F531 PerfeCTa SYBR Green FastMix Quanta Bio 95072 Bovine Serum Albumin (BSA) BioShop 005PJD Bovine Serum Albumin (BSA) Jackson Immunoresearch 005PEF Hoechst 33258 Sigma Aldrich 94403 (Continued on next page) Cell Reports 35, 109226, June 8, 2021 e1
Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control, Mutagenesis
R&D Systems is a verified supplier 
R&D Systems manufactures this product 
