Journal: Cell Death & Disease

Article Title: HUMANIN produced by human efferocytic macrophages promotes the resolution of inflammation

doi: 10.1038/s41419-025-07909-1

Figure Lengend Snippet: A Peritonitis was induced in C57/Bl6 mice by intraperitoneal (i.p.) injection of 1 mg zymosan-A (Zy.) resuspended in PBS (open dark blue circles) or vehicle PBS (Veh., black circles). HUMANIN (Hu, 20 µg in PBS) or vehicle (Veh) was injected i.p. 30 min before zymosan-A (Zy.+Hu., open light blue circles vs . Hu.+Veh., open circles, respectively). Peritoneal washes were performed at different time points (6 h,12 h 24 and 72 h) and cells were used for different analyses. B Western blot analysis of mouse Humanin (HN) in cell-free peritoneal fluid at 6 h, 12 h, 24 h and 72 h after zymosan-A (Zy.) injection. Total protein obtained with stain-free imaging was used as a control. ( n = 3). C Total cell count in peritoneal fluid was performed manually using Trypan blue staining. D Flow cytometry was used to assess number of neutrophils Ly6G + , F4/80 − ) and myeloid cells (Ly6G − , F4/80 low/High ) contained in the peritoneal fluid at the different indicated time points. E Neutrophil viability in the peritoneal fluid at the indicated time points was assessed by flow cytometry using Annexin V and 7-AAD. F After zymosan-A injection, the peritoneal cavity of the mice, treated with HUMANIN or not, was flushed at the indicated time points with PBS and TNFα, IL-6 and IL-1β levels in the recovered fluids were measured by ELISA. Statistics: Two-way ANOVA and two-tailed paired t-test were used according to test requirements. Results are displayed as mean ± SEM ( n = 10–14 mice), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Lysis was stopped by PBS addition and cells were centrifuged and resuspended in complete RPMI medium (GIBCO).

Techniques: Injection, Western Blot, Staining, Imaging, Control, Cell Counting, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Schematic for the purification and transcriptomic profiling of P-bodies from HEK293T cells based on the expression of GFP-LSM14A. (b) Representative IF imaging of GFP-LSM14A puncta (green), colocalizing with EDC4 puncta (red) in HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10μm). n=3 independent experiments. (c) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in HEK293T cells. (d) Representative imaging of GFP-LSM14A puncta (green) in control and DDX6 KO HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in control (n=50 cells) and DDX6 KO (n=50 cells) HEK293T cells (right panel). Unpaired two-sided Student’s t-test, mean ± s.d., ****: p<0.0001. (e) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in control and DDX6 KO HEK293T cells. (f) MA plot of RNA-seq data depicting P-body enriched genes in red and cytoplasm enriched genes in blue in HEK293T cells (n=2 biological independent samples per group, p<0.05). (g) GO pathway analysis of P-body enriched mRNAs in HEK293T cells, using expressed genes as background. Enrichment was tested by two-sided Fisher’s exact test with multiple testing correction (Benjamini–Hochberg FDR). (h) GO pathway analysis of cytoplasm enriched mRNAs in HEK293T cells, using expressed genes as background. Enrichment was tested by two-sided Fisher’s exact test with multiple testing correction (Benjamini–Hochberg FDR). (i) Representative FISH imaging of POLK RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green). Nuclei were counterstained with DAPI (blue) (scale: 10μm). n=3 independent experiments. (j) Quantification of POLK mRNA molecules in P-bodies based on FISH (n=50 cells; right) and P-body sequencing (right), mean ± s.d. (k) Read coverage distribution over the gene body of the longest annotated isoforms for genes enriched in P-bodies or cytoplasm in HEK293T cells. (l) PolyA tail length as determined in 51 compared to P-body enrichment based on SMART-Seq and SnapTotal-Seq, Pearson correlation test. (m) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in HEK293T cells, Pearson correlation test (two sided), p=3.94e-98.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Purification, Expressing, Imaging, Flow Cytometry, Control, RNA Sequencing, Sequencing

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Schematic of the conversion from naïve to primed state mouse ES cells (left panel). Representative bright field images of naïve and primed mouse ES cells (scale: 50μm) (right panel). n=3 independent experiments. (b) Heatmap showing expression levels of naïve-specific and primed-specific transcripts. (n=2 biological independent samples per group). (c) Representative IF imaging of EDC4 puncta (green) in naïve and primed mouse ES cells. Cell membranes were labeled with Phalloidin (red) and nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in naïve (n=60 cells) and primed (n=60 cells) mouse ES cells (right panel). Unpaired two-sided Student’s t-test, mean ± s.d., n.s.: p >0.05, p=0.0843. (d) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in naïve and primed mouse ES cells. (e) Heatmap showing expression levels of differentially enriched mRNAs between purified P-body fractions of naïve and primed mouse ES cells, with GO pathway analysis using expressed genes as a background for P-body-enriched transcripts for the indicated clusters. Clusters were generated manually to represent genes that are P-body enriched in both naive and primed mouse ES cells, or specifically enriched in one cell type. Gene number in each transcript cluster is indicated in the figure (n=2 biological independent samples per group, p<0.05). (f) GSEA analysis of blastomere-related genes 64 in purified P-body fraction vs. cytoplasmic fraction from naïve mouse ES cells, NES=1.28, p=0.03. Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method. (g) A schematic of the strategy for P-body dissolution in mouse naïve ES cells carrying blastomere-specific reporters (MERVL-tdTomato and ZSCAN4-GFP). (h, i) Flow cytometric quantification of MERVL-tdTomato + and ZSCAN4-GFP + cells upon Lsm14a , Ddx6, and Eif4enif1 KO in mouse naïve ES cells. Control, Lsm14a , Ddx6 KO and Eif4enif1 KO (n=3 biological independent samples per group). (j, k) RNA-seq data from sorted MERVL/ZSCAN4-negative and -positive mouse ES cells after Ddx6 KD, showing expression levels of pluripotency- and totipotency-related transcripts (j) and transposable elements (k).

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Expressing, Imaging, Labeling, Flow Cytometry, Purification, Generated, Dissolution, Control, RNA Sequencing

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in naïve and primed mouse ES cells. Ribosome profiling data from 69 , Pearson correlation test (two sided). (b) A schematic showing CRISPR-Cas9-based homozygous insertion of FKBP12F36V-HA-P2A-mCherry sequence in place of the stop codon of the endogenous Ddx6 allele. (c) Representative intracellular flow cytometry plots for DDX6 in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. (d) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. Nuclei were counterstained with DAPI (blue) (scale: 10μm). (e) P-body number in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. DMSO (n=70 cells), 3 hour-dTAG13 (n=70 cells), 6 hour-dTAG13 (n=70 cells), 9 hour-dTAG13 (n=70 cells), unpaired two-sided Student’s t-test, mean ± s.d., ****: p <0.0001. (f) Cumulative distribution function (CDF) plot showing ribosome occupancy (log2 FC) of P-body enriched and P-body-depleted mRNAs for untreated (DMSO) vs. dTAG-13 treated (6hrs) Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, Wilcox test p=2.97e-129. (g) Box plots showing the change in ribosome occupancy (log2(ribosome bound/total RNA) in the degron-log2(ribosome bound/total RNA) in the control) of P-body enriched genes versus all other genes. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Statistical significance was assessed by unpaired two sided t-test, mean ± s.d., ****: p<0.0001; padj =1.6e-38 (Holm’s method); all other genes n=9133, P-body n=2843. (h) Normalized Enrichment Score (NES) of gene sets from (2C) 64 p=0.003, (Naïve) 105 p=0.923, and (Primed) 70 p=0.499, Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method (FDR<0.05). (i) Box plots showing the change in ribosome occupancy of P-body enriched 2C-related genes 64 compared to non-P-body enriched 2C genes. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5 IQR. Unpaired two-sided t-test, mean ± s.d, **: p<0.01; padj=0.044 (Holm’s method); all other genes n=254, P-body n=104. (j) Box plots showing the change in protein levels log2(degron/ctrl) of all P-body enriched genes compared to P-body depleted genes (cytoplasm), after 1 day and 3 days of dTAG-13 treatment. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Unpaired two-sided t-test, mean ± s.d, ****: p<0.0001; padj=8.5e-7, padj=6.6e-6 (Holm’s method); cytoplasm n=1090, P-body n=1661. (k) Box plots showing the change in protein levels log2(degron/ctrl) of P-body enriched 2C-related genes compared to P-body depleted genes (cytoplasm), after 1 day and 3 days of dTAG-13 treatment. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Unpaired two-sided t-test, mean ± s.d, n.s.: p>0.05, *:p<0.05; padj=0.086, padj=0.047 (Holm’s method); cytoplasm n=28, P-body n=42. (l) Heatmap showing protein levels of 2C-related genes after 1 day and 3 days of dTAG-13 treatment compared to control samples.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Activation Assay, Gene Expression, RNA Sequencing, CRISPR, Sequencing, Flow Cytometry, Imaging, Control

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) A schematic of the strategy for miR-300 inhibition in mouse naïve ES cells. (b) qRT-PCR analysis of the expression for miR-300 targets in P-bodies of mouse naïve ES cells after miR-300 inhibition and for control, n=3 biological independent samples per group, unpaired two-sided Student’s t-test, mean ± s.d., *: p<0.05, **: p<0.01, ****: p<0.0001, p=0.0009, p=0.0133, p=0.0237, p=0.0195, p=0.0009, p=0.0004, p=0.0002. (c) Heatmap showing expression levels of differentially enriched 2C-related genes in mouse naïve ES cells after miR-300 inhibition and for control. (d) Representative IF imaging of EDC4 puncta (green), and Nanog-MS2 (red) in Nanog Let7 wt (upper panel) and Nanog Let7 mut cells (lower panel). Nuclei were counterstained with DAPI (blue) (scale: 10μm). n=3 independent experiments. (e) qRT-PCR analysis of Nanog expression in Nanog Let7 wt and Nanog Let7 mut cells compared to CTRL cells (Nanog KO). n=3 biologically independent samples per group, mean ± s.d. (f) Representative western blot showing NANOG protein levels in Nanog Let7 wt and Nanog Let7 mut cells compared to CTRL cells ( Nanog KO). (g-i) Representative pictures (g) and quantification of Alkaline Phosphatase staining of cell colony number (h) and size (i) from Nanog Let7 wt and Nanog Let7 mut cells cultured in FBS+LIF conditions. Unpaired two-sided Student’s t-test, n=3 biologically independent samples per group, mean ± s.d., *: p<0.05, ***: p<0.001, p=0.0002, p=0.0135. (j) Heatmap showing expression levels of differentially enriched 8C-related mRNAs between purified P-body and cytoplasmic fractions in human naïve ES cells. (n=2 biological independent samples per group). (k) A schematic of the strategy for P-body dissolution in naïve human ES cells carrying blastomere-specific reporter (TPRX1-GFP). Flow cytometric analysis of TPRX1-GFP + expression after four days of DDX6 KD. (l) Quantification of TPRX1-GFP + cells upon DDX6 KD. Unpaired two-sided Student’s t-test, n=4 biologically independent samples per group, mean ± s.d., ****: p<0.0001. (m) Representative IF imaging of H3Y1 (green) positive cells in naïve human ES cells upon DDX6 KO compared to control cells. Nuclei were counterstained with DAPI (blue) (scale: 50μm) (Left panel). Quantification of H3Y1 + cells upon DDX6 suppression. Unpaired two-sided Student’s t-test, sgControl (n=5 fields), sgDDX6 (n=8 fields), mean ± s.d., ****: p<0.0001 (Right panel). (n, o) RNA-seq data from primed, naïve and TPRX1 positive human ES cells following DDX6 KD, showing expression levels of pluripotency- and totipotency-related transcripts (n) and transposable elements (o). (p) GSEA analysis for hPGCLC-related gene expression signature 97 in P-body vs cytoplasm differential expression in human primed ES cells. Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method. (q) A schematic of hiPSC to PGCLC differentiation. HiMeLCs: human incipient mesoderm-like cells. (r) Flow cytometric analysis of TFAP2C-GFP and BLIMP1-TOMATO expression after four days of PGCLC differentiation in 3D aggregates (left). Quantification of TFAP2C-GFP + /BLIMP1-TOMATO + cells by flow cytometry for three experiments (right). Error bars indicate mean ± s.d., n=3 biologically independent samples per group, statistical significance was determined using a two-sided unpaired Student’s t-test, ***: p<0.001, p=0.0003. (s) RNA-seq analysis of human iPS cells (BTAG cell line 97 ), intermediate mesenchymal-like cells (iMeLCs), and TFAP2C/BLIMP1-negative and -positive cells following DDX6 suppression, showing expression levels of pluripotency, mesenchymal, and PGC-related genes.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Imaging, Western Blot, Staining, Cell Culture, Purification, Dissolution, RNA Sequencing, Gene Expression, Quantitative Proteomics, Flow Cytometry

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Representative image of pre-sorted cell lysate containing GFP-LSM14A + P-bodies (scale: 10μm). n=3 independent experiments. (b) Representative imaging of GFP-LSM14A puncta (green) in control and DDX6 KD HEK293T cells. Nuclei were counterstained with DAPI (blue) (scale: 10μm) (left panel). P-body number in control (n=50 cells) and DDX6 KD (n=50 cells) HEK293T cells (right panel). Unpaired two-sided Student’s t-test, mean ± s.d., ****: p<0.0001. (c) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in control and DDX6 KD HEK293T cells. (d) Representative flow cytometry plots showing gating for SYTOX BLUE + events in GFP + and GFP − gates of pre-sorted cytoplasmic fraction from HEK293T cells. (e) Venn diagram showing the overlap between P-body-associated mRNAs in HEK293T from SMART-Seq and SnapTotal-Seq. (f) Venn diagram showing the overlap between P-body-associated mRNAs in HEK293T of this study (blue) and a published dataset from 29 (grey). (g) Representative FISH imaging of TET2 RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green). Nuclei were counterstained with DAPI (blue) (scale: 5 μm). Quantification of TET2 mRNA molecules in P-bodies based on FISH (n=30 cells; left) and P-body sequencing (right). (h) Correlation between mRNA half-life as determined in ref. 50 and P-body enrichment in HEK293T cells, Pearson correlation test (two-sided), p=3.08e-106. (i) Length (left panel) and GC content (right panel) density plots of mRNAs in purified P-body and cytoplasmic fractions of HEK293T cells.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Imaging, Control, Flow Cytometry, Sequencing, Purification

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a, b) Representative FISH imaging of OCT4 RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green) in primed ES cells (a) and endoderm progenitors (b) with corresponding quantification (n=30 cells; right). Nuclei were counterstained with DAPI (blue) (scale: 5μm). (c) GSEA of P-body vs cytoplasm differential expression in human primed ES cells, endoderm progenitors, and mesoderm progenitors, with gene sets related to the differentiated and preceding developmental stage. (d) A schematic of the strategy for P-body dissolution in human endoderm progenitors during their differentiation to hepatocytes. (e) Representative IF imaging of AFP (green) positive cells in hPSCs upon DDX6 KO compared to control cells. Nuclei were counterstained with DAPI (blue) (scale: 100μm) (Left panel). Quantification of AFP + cells upon DDX6 suppression. Unpaired two-sided Student’s t-test, sgControl (n=3 fields), sgDDX6 (n=3 fields), mean ± s.d., ****: p<0.0001 (Right panel). (f) qRT-PCR analysis for the indicated genes in hepatocytes. Unpaired two-sided Student’s t-test, mean ± s.d., n=3 biologically independent samples per group,**: p<0.01, ****: p<0.0001; p=0.0013, p=0.0071, p=0.0004. (g) A schematic of neuron maturation. (h) Venn diagram showing the overlap between P-body-associated mRNAs in neurons cultured for 7 and 20 days. (i) GSEA of P-body vs cytoplasm differential expression in day 7 and day 20 neurons, with a neural progenitor related gene sets. Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method. (j) A schematic of endoderm progenitor maturation. (k) GSEA of P-body vs cytoplasm differential expression in endoderm progenitors and mature endoderm progenitors with a gene set for primed human ES cell-related genes. Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method. (l) GSEA of P-body vs cytoplasm differential expression in naïve human ES cells with a gene set for 8-cell embryo 59 . Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Imaging, Quantitative Proteomics, Dissolution, Control, Quantitative RT-PCR, Cell Culture

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) GSEA for blastomere-related transcripts 64 in P-body vs cytoplasm differential enrichment data from mouse ES cells using SnapTotal-Seq. Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method. (b, c) Representative FISH imaging of Dppa2 RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green) in naïve (b) and primed (c) ES cells with corresponding quantification (n=30 cells; right). Nuclei were counterstained with DAPI (blue) (scale: 5μm). (d, e) Representative FISH imaging of Rex1 ( Zfp42 ) RNA molecules (red) combined with imaging of GFP-LSM14A puncta (green) in naïve (d) and primed (e) ES cells with corresponding quantification (n=30 cells; right). Nuclei were counterstained with DAPI (blue) (scale: 5μm). (f) Cell cycle analysis of MERVL-negative and -positive cells after Ddx6 KD. n=3 biologically independent samples per group, mean ± s.d. (g) Venn diagram showing the overlap between P-body-associated mRNAs in ES cells from SMART-Seq and SnapTotal-Seq. (h) mRNA half-life as determined in 67 compared to P-body enrichment in primed mouse ES cells, 2C genes 64 are highlighted in pink, Pearson correlation test (two-sided). (i) Read coverage distribution over the gene body of the longest annotated isoforms of genes enriched in P-bodies in naïve mouse ES cells using SMART-Seq and SnapTotal-Seq. (j) Poly-A tail length as determined in 68 compared to P-body enrichment based on SMART-Seq and SnapTotal-Seq, Pearson correlation test (two-sided), p=2.35e-69.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Imaging, Cell Cycle Assay

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Representative western blot showing DDX6 protein levels in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. n=3 independent experiments. (b) Representative flow cytometry plots showing gating for GFP-LSM14A + P-bodies in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. (c) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, treated with dTAG-13 for 3 and 9 hours. Nuclei were counterstained with DAPI (blue) (scale: 10mm). n=3 independent experiments. (d) GO terms for P-body enriched genes with increased ribosome occupancy of Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours). Enrichment was tested by two-sided Fisher’s exact test with multiple testing correction (Benjamini–Hochberg method). (e) Volcano plot of RNA-seq data depicting differential expression of total RNA fraction in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours, with P-body enriched genes in red and cytoplasm enriched genes in blue (n=3 biologically independent samples per group, p < 0.05). (f) GO terms of P-body enriched genes that showed downregulated gene expression in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells following dTAG13 treatment for 6 hours. Enrichment was tested by two-sided Fisher’s exact test with multiple testing correction (Benjamini–Hochberg method).

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: Disruption, Western Blot, Flow Cytometry, Imaging, RNA Sequencing, Quantitative Proteomics, Gene Expression