Journal: Nature Communications
Article Title: Phase separated condensates of ATRX regulate neural progenitor identity
doi: 10.1038/s41467-025-61881-0
Figure Lengend Snippet: a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA from ΔEx15 hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human iPSC-derived NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Article Snippet: The human iPSC control lines (1210B2 and RPC771 (ReproCELL)) and the genome-edited derivative of RPC771 (ΔEx15) were maintained as previously described , .
Techniques: Expressing, Mutagenesis, Construct, Sequencing, Immunostaining, Two Tailed Test, Gene Expression, Derivative Assay, Staining
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![a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA <t>from</t> <t>ΔEx15</t> hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human <t>iPSC-derived</t> NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9971/pmc12259971/pmc12259971__41467_2025_61881_Fig5_HTML.jpg)