Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) HCT116 cells stably expressing DDX6-GFP were plated in 96-well plates, treated with 280 compounds at 10µM concentrations, and subjected to high-content imaging using the CQ1 confocal quantitative imaging system. (b) The analyzed images consist of four channels: (i) Bright-field image for cellular morphology, (ii) Mitochondrial network, (iii) Processing body, and (iv) Nucleus. Merged composite demonstrates spatial relationships between these subcellular compartments. Scale bar: 10μm. (c) Mitochondrial channels were processed through Cellpose 3.0 to generate a curated dataset containing over 400,000 high-quality single-cell images. (d) A contrastive clustering framework was implemented for unsupervised feature extraction, followed by UMAP dimensionality reduction to identify compounds with analogous mechanism-of-action (MOA) profiles through cluster localization analysis. (e) Quantitative analysis of P-body formation followed by drug treatment. (f) Mechanistic evaluation of lead compounds via imaging analysis.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Stable Transfection, Expressing, Imaging, Extraction

Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) A simulation model of intracellular p-body was constructed to generate synthetic p-body distributions with ground truth annotations. (b) A YOLO-v7 architecture trained on synthetic datasets was implemented for automated identification and quantitative analysis of P-body formation. (c) Example of P-body detection, achieving >95% agreement with manual annotations . (d) P-body numbers per cell in the time course under different drug treatment groups. (e) DDX6-GFP expression level (a.u.) per cell under different drug treatment groups. Error bars represent the STD of three independent analyses for d and e. (f) Quantitative analysis of P-body numbers at 6 hours post-treatment across different drug groups. (g) Quantitative analysis of DDX6 expression level (a.u.) at 6 hours post-treatment across different drug groups. The p -values were determined using the two-tailed Mann–Whitney test for f and g. The statistical significance compared with DMSO was indicated as *** P < 0.001; ** P < 0.01; * P < 0.05; ns, no significant difference. Data points that lay outside the 15% - 85% range were deemed outliers and excluded from the statistical analysis. (h, i) Mechanism of Action (MOA) profiling for drugs in Groups 1 and 3.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Construct, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) HCT116 cells were knocked down using JAK1 and JAK2 siRNA, and immunostained for P-body components DDX6 (magenta) and EDC4 (green). The nuclei were visualized with DAPI (blue). Scale bar, 10μm. (b) Quantification of P-body number per cell across three experimental groups. Statistical significance determined by an unpaired t-test was indicated as *** P < 0.001. (c) Model of JAK/STAT signaling pathway-mediated P-body regulation. JAK is activated when cytokines or growth factors bind to their respective receptors, leading to receptor dimerization, JAK and STAT phosphorylation, and subsequent transcriptional regulation. Inhibition of the pathway by knockdown of JAK1/2 leads induction of P-body formation. The JAK inhibitors identified in this work that modulate P-body formation are shown in the right panel.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Phospho-proteomics, Inhibition, Knockdown

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Schematic of the IP-MS protocol. (B) Western blot of immunoprecipitation experiments. (C) GO molecular function analysis of DDX6 interactors (FC>1.5). (D) GO cellular component analysis of DDX6 interactors (FC>1.5). (E) DDX6 IP-MS data, n=3, unpaired Student’s t-test, FC>1.5; P<0.05. (F) Heatmap showing protein expression changes determined by MS. (G) Flow cytometric quantification of OCT4-GFP+ hESCs in mTeSR1 and mTeSR1 lacking bFGF and TGFβ. (H) Schematic of DDX6 protein with E247Q mutation (red square) in the helicase domain (blue square) (upper panel). QRT-PCR analysis of DDX6 expression (lower panel). (I) Immunofluorescence image showing protein expression of DDX6 (scale: 10μm) and EDC4 (scale: 10μm). (J) Immunofluorescence image showing protein expression of NANOG (scale: 100μm). (K) QRT-PCR analysis of selected pluripotency genes. (L) QRT-PCR analysis of selected pluripotency genes. (M) Immunofluorescence image showing protein expression of DDX6 (scale: 50μm, inset 2X) and EDC4 (scale: 50μm, inset 2X) in sgCTRL, sgDDX6 #5 hiPSCs treated with dox for 1 weeks and sgDDX6 #5 “Wash Out” (WO) which have been treated with dox for 1 week followed by 7 days of dox withdrawal. (N) QRT-PCR analysis of selected pluripotency genes.

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques: Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Quantitative RT-PCR, Immunofluorescence

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Immunofluorescence image showing protein expression of EDC4 (scale: 50μm, inset 2X) in control and DDX6 overexpressing hESCs (left panel). P-body counts per cell (right panel), n=6, mean ± s.d. (B) Flow cytometric quantification of OCT4-GFP+ control (n=3) and DDX6 overexpressing (n=6) hESCs cultured in mTeSR1 and mTeSR1 supplemented with TGFβi. (C) Heatmap showing differentially expressed genes (FC>1.5; FDR<0.001) in control and DDX6 overexpressing hiPSCs cultured in mTeSR1. (D) Heatmap showing differentially expressed genes (FC>1.5; FDR<0.001) in control and DDX6 overexpressing hiPSCs cultured in mTeSR1 supplemented with TGFβi. (E) Schematic of the eCLIP-seq protocol. (F) Histogram of region-based fold change (FC) for DDX6 eCLIP-seq read density over size-matched input (FC>2; P<0.001). (G) GO analysis of DDX6 targets in hiPSCs (FC>2; P<0.001). (H) Venn diagram showing overlap for DDX6 eCLIP-seq targets (FC>2; P<0.001) and P-body-enriched mRNAs (Hubstenberger et al., 2017). (I) Polysome profile. (J) Cumulative distribution function (CDF) plot showing translation rate fold (log2) change (FC) of P-body enriched DDX6-target and non-target mRNAs for sgDDX6 #5 vs sgCTRL hiPSCs. Statistical significance was calculated using the Mann–Whitney U test. (K) Violin plots showing the Polysome/Input RPKM values for the indicated transcripts (n=3 each condition). (L) Violin plots showing expression values for the indicated proteins (n=3 each condition). See also Figure S6 and Table S3.

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques: Immunofluorescence, Expressing, Cell Culture, MANN-WHITNEY

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Schematic of dCas9-KRAB and sgRNA vectors and genomic positions of the sgRNA targeting the DDX6 TSS (upper panel). QRT-PCR analysis of DDX6 in sgCTRL and sgDDX6 #5 cells treated with dox. Unpaired Student’s t test. n=3, mean ± s.d., ****P<0.0001. (B) Immunofluorescence image showing protein expression of DDX6 (scale: 50 μm; inset 2X). (C) Immunofluorescence image showing protein expression of EDC4 (scale: 50 μm; inset 2X) (left panel). P-body count per cell (right panel), n=6, mean ± s.d. (D) Schematic of hiPSCs differentiation (upper panel). FACS analysis of the proportion of NANOG+ cells (lower panel). (E) Immunofluorescence images showing protein expression of NANOG (scale: 100μm). (F) MA plots of RNA-seq data depicting upregulated genes in red and downregulated genes in blue (FC>1.5; FDR<0.01). (G) GO and KEGG pathways analysis of upregulated genes (FC>1.5; FDR<0.01) in sgDDX6 #5 vs sgCTRL cells. (H) Hierarchical clustering of RNA-seq samples. (I) Heatmap showing expression levels of selected pluripotency genes (n=2 each condition).

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques: Quantitative RT-PCR, Immunofluorescence, Expressing, RNA Sequencing Assay

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Scatter plot showing correlation of ATAC-seq data for sgCTRL (n=2) and sgDDX6 #5 (n=2) hiPSCs. Blue dots indicate genomic regions showing significantly decreased chromatin accessibility in DDX6 depleted cells (>1.5-fold change, P-value<0.001; n=3999); red dots indicate genomic regions showing significantly increased chromatin accessibility in DDX6 depleted cells (1.5-fold change, P-value<0.001; n=7420). (B) TF motif enrichment on sgDDX6 gained and lost ATAC-seq peaks. (C) Scatter plot showing H3K27ac ChIP-seq data for sgDDX6 #5 (n=2) and sgCTRL (n=2) hiPSCs. Red dots indicate genomic regions with significant decreased H3K27ac signal in DDX6 depleted cells (>2-fold change; n=712); green dots indicate genomic regions with significant increased H3K27ac signal in DDX6 depleted cells (2-fold change; n=3528). (D) H3K27ac signal at pluripotency-specific super-enhancers (n=684) in sgCTRL (n=2) and sgDDX6 (n=2) hiPSCs. Statistical significance was determined using a Student’s t-test. (E) Gene tracks of individual genes based on RNA-seq, ChIP-seq and ATAC-seq data. (F) Scatter plot showing H3K9me3 ChIP-seq data for sgCTRL (n=2) and sgDDX6 #5 (n=2) hiPSCs. Red dots indicate genomic regions showing significantly decreased H3K9me3 coverage in DDX6 depleted cells (>2-fold change; n=1494); green dots indicate genomic regions with significantly increased H3K9me3 signal in DDX6 depleted cells (2-fold change; n=1279). (G) Scatter plot showing correlation of ATAC-seq data for shCTRL- (n=2) and shDDX6-infected (n=2) human myoblasts. Blue dots indicate genomic regions with significantly decreased chromatin accessibility in DDX6 depleted cells (>1.5-fold change, P-value<0.001; n=1099); red dots indicate genomic regions with significantly increased chromatin accessibility in DDX6 depleted cells (1.5-fold change, P-value<0.001; n=1864). (H) Heatmaps showing enrichment of the indicated histone modifications for regions that gained and lost ATAC-seq peaks in shDDX6 myoblasts relative to control. (I) TF motif enrichment for regions that gained and lost ATAC-seq peaks in shDDX6 myoblasts relative to control. (J) Violin plots showing the Polysome/Input RPKM values for KDM4B (n=3 each condition) in hiPSCs. (K) KDM4B mRNA (n=2, mean ± s.d.) and protein expression levels in hiPSCs (n=3, mean ± s.d.), unpaired Student’s t-test, **P<0.01. (L) Immunofluorescence images showing MyHC protein expression (left panel). Quantification of MyHC+ cells (right panel). n=4, mean ± s.d., unpaired Student’s t-test, **P<0.01 (scale: 100μm, left panel). (M) QRT-PCR analysis for the indicated genes in differentiating myoblast cultures. n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01, ***P<0.001. (N) Flow cytometric quantification of OCT4-GFP+ hESCs infected with the empty retroviral vector PCLP or PCLP-KDM4B and cultured in mTeSR1 and mTeSR1 lacking bFGF and TGFβ.

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques: ChIP-sequencing, RNA Sequencing Assay, Infection, Expressing, Immunofluorescence, Quantitative RT-PCR, Plasmid Preparation, Cell Culture

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Gene tracks showing RNA-seq data. (B) Single cell RNA-seq data for DDX6 expression in human preimplantation embryos (Petropoulos et al., 2016). Epi: Epiblast; Pe: Primitive Endoderm; TE: trophectoderm. (C) RNA-seq and protein expression data for DDX6 in primed and naïve hESCs (Di Stefano et al., 2018). For RNA-seq data, n=5, mean ± s.d., unpaired Student’s t-test, ***P<0.001. For proteomic data, n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01. (D) Analysis of repetitive element expression. Repeats with significant expression differences are indicated in red (FC>1.5, FDR <0.05). (E) Differentially methylated promoter regions in DDX6 depleted cells relative to control cells. Significantly hypomethylated promoters are shown in red (>10% difference, P<0.01); significantly hypermethylated promoters are shown in blue (>10% difference, P<0.01). (F) PCA analysis of RNA-seq data for the indicated samples based on differentially expressed genes between shDDX6 #1 and shCTRL hESCs. (G) Flow cytometric detection of ΔPE OCT4-GFP+ cells after reversion of primed hESCs to a naïve state in 5i/LAF medium. Black curve shows the negative control. (H) QRT-PCR analysis for the indicated genes after 8 days of 5i/LAF treatment. Values are represented respect to control cells at day 0. n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques: RNA Sequencing Assay, Expressing, Methylation, Negative Control, Quantitative RT-PCR

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Summary of phenotypes in DDX6 depleted stem cell populations. (B) Model proposing how DDX6 impacts cell fate through modulation of P-body homeostasis.

Article Snippet: The following antibodies were used for Western blot: βIII-TUBULIN (1:2000, Cell Signaling Technology, clone 9F3, cat. #2128); Histone H3 (1:10,000, AbCam, cat. #1791); DDX6 (1:2000, Novus Biologicals, NB200–192); β-ACTIN (1:2000, Cell Signaling Technology, clone 13E5, cat. #4970); PABP (1:500, Santa Cruz Biotechnology, clone 10E10, cat. #SC-32318), ATXN2L (1:100, Bethyl Laboratories, cat. #A301–370A), DCP1B (1:1000, Cell Signaling Technology, clone D2P9W, cat. #13233).

Techniques:

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Schematic of the IP-MS protocol. (B) Western blot of immunoprecipitation experiments. (C) GO molecular function analysis of DDX6 interactors (FC>1.5). (D) GO cellular component analysis of DDX6 interactors (FC>1.5). (E) DDX6 IP-MS data, n=3, unpaired Student’s t-test, FC>1.5; P<0.05. (F) Heatmap showing protein expression changes determined by MS. (G) Flow cytometric quantification of OCT4-GFP+ hESCs in mTeSR1 and mTeSR1 lacking bFGF and TGFβ. (H) Schematic of DDX6 protein with E247Q mutation (red square) in the helicase domain (blue square) (upper panel). QRT-PCR analysis of DDX6 expression (lower panel). (I) Immunofluorescence image showing protein expression of DDX6 (scale: 10μm) and EDC4 (scale: 10μm). (J) Immunofluorescence image showing protein expression of NANOG (scale: 100μm). (K) QRT-PCR analysis of selected pluripotency genes. (L) QRT-PCR analysis of selected pluripotency genes. (M) Immunofluorescence image showing protein expression of DDX6 (scale: 50μm, inset 2X) and EDC4 (scale: 50μm, inset 2X) in sgCTRL, sgDDX6 #5 hiPSCs treated with dox for 1 weeks and sgDDX6 #5 “Wash Out” (WO) which have been treated with dox for 1 week followed by 7 days of dox withdrawal. (N) QRT-PCR analysis of selected pluripotency genes.

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques: Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Quantitative RT-PCR, Immunofluorescence

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Immunofluorescence image showing protein expression of EDC4 (scale: 50μm, inset 2X) in control and DDX6 overexpressing hESCs (left panel). P-body counts per cell (right panel), n=6, mean ± s.d. (B) Flow cytometric quantification of OCT4-GFP+ control (n=3) and DDX6 overexpressing (n=6) hESCs cultured in mTeSR1 and mTeSR1 supplemented with TGFβi. (C) Heatmap showing differentially expressed genes (FC>1.5; FDR<0.001) in control and DDX6 overexpressing hiPSCs cultured in mTeSR1. (D) Heatmap showing differentially expressed genes (FC>1.5; FDR<0.001) in control and DDX6 overexpressing hiPSCs cultured in mTeSR1 supplemented with TGFβi. (E) Schematic of the eCLIP-seq protocol. (F) Histogram of region-based fold change (FC) for DDX6 eCLIP-seq read density over size-matched input (FC>2; P<0.001). (G) GO analysis of DDX6 targets in hiPSCs (FC>2; P<0.001). (H) Venn diagram showing overlap for DDX6 eCLIP-seq targets (FC>2; P<0.001) and P-body-enriched mRNAs (Hubstenberger et al., 2017). (I) Polysome profile. (J) Cumulative distribution function (CDF) plot showing translation rate fold (log2) change (FC) of P-body enriched DDX6-target and non-target mRNAs for sgDDX6 #5 vs sgCTRL hiPSCs. Statistical significance was calculated using the Mann–Whitney U test. (K) Violin plots showing the Polysome/Input RPKM values for the indicated transcripts (n=3 each condition). (L) Violin plots showing expression values for the indicated proteins (n=3 each condition). See also Figure S6 and Table S3.

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques: Immunofluorescence, Expressing, Cell Culture, MANN-WHITNEY

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Schematic of dCas9-KRAB and sgRNA vectors and genomic positions of the sgRNA targeting the DDX6 TSS (upper panel). QRT-PCR analysis of DDX6 in sgCTRL and sgDDX6 #5 cells treated with dox. Unpaired Student’s t test. n=3, mean ± s.d., ****P<0.0001. (B) Immunofluorescence image showing protein expression of DDX6 (scale: 50 μm; inset 2X). (C) Immunofluorescence image showing protein expression of EDC4 (scale: 50 μm; inset 2X) (left panel). P-body count per cell (right panel), n=6, mean ± s.d. (D) Schematic of hiPSCs differentiation (upper panel). FACS analysis of the proportion of NANOG+ cells (lower panel). (E) Immunofluorescence images showing protein expression of NANOG (scale: 100μm). (F) MA plots of RNA-seq data depicting upregulated genes in red and downregulated genes in blue (FC>1.5; FDR<0.01). (G) GO and KEGG pathways analysis of upregulated genes (FC>1.5; FDR<0.01) in sgDDX6 #5 vs sgCTRL cells. (H) Hierarchical clustering of RNA-seq samples. (I) Heatmap showing expression levels of selected pluripotency genes (n=2 each condition).

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques: Quantitative RT-PCR, Immunofluorescence, Expressing, RNA Sequencing Assay

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Scatter plot showing correlation of ATAC-seq data for sgCTRL (n=2) and sgDDX6 #5 (n=2) hiPSCs. Blue dots indicate genomic regions showing significantly decreased chromatin accessibility in DDX6 depleted cells (>1.5-fold change, P-value<0.001; n=3999); red dots indicate genomic regions showing significantly increased chromatin accessibility in DDX6 depleted cells (1.5-fold change, P-value<0.001; n=7420). (B) TF motif enrichment on sgDDX6 gained and lost ATAC-seq peaks. (C) Scatter plot showing H3K27ac ChIP-seq data for sgDDX6 #5 (n=2) and sgCTRL (n=2) hiPSCs. Red dots indicate genomic regions with significant decreased H3K27ac signal in DDX6 depleted cells (>2-fold change; n=712); green dots indicate genomic regions with significant increased H3K27ac signal in DDX6 depleted cells (2-fold change; n=3528). (D) H3K27ac signal at pluripotency-specific super-enhancers (n=684) in sgCTRL (n=2) and sgDDX6 (n=2) hiPSCs. Statistical significance was determined using a Student’s t-test. (E) Gene tracks of individual genes based on RNA-seq, ChIP-seq and ATAC-seq data. (F) Scatter plot showing H3K9me3 ChIP-seq data for sgCTRL (n=2) and sgDDX6 #5 (n=2) hiPSCs. Red dots indicate genomic regions showing significantly decreased H3K9me3 coverage in DDX6 depleted cells (>2-fold change; n=1494); green dots indicate genomic regions with significantly increased H3K9me3 signal in DDX6 depleted cells (2-fold change; n=1279). (G) Scatter plot showing correlation of ATAC-seq data for shCTRL- (n=2) and shDDX6-infected (n=2) human myoblasts. Blue dots indicate genomic regions with significantly decreased chromatin accessibility in DDX6 depleted cells (>1.5-fold change, P-value<0.001; n=1099); red dots indicate genomic regions with significantly increased chromatin accessibility in DDX6 depleted cells (1.5-fold change, P-value<0.001; n=1864). (H) Heatmaps showing enrichment of the indicated histone modifications for regions that gained and lost ATAC-seq peaks in shDDX6 myoblasts relative to control. (I) TF motif enrichment for regions that gained and lost ATAC-seq peaks in shDDX6 myoblasts relative to control. (J) Violin plots showing the Polysome/Input RPKM values for KDM4B (n=3 each condition) in hiPSCs. (K) KDM4B mRNA (n=2, mean ± s.d.) and protein expression levels in hiPSCs (n=3, mean ± s.d.), unpaired Student’s t-test, **P<0.01. (L) Immunofluorescence images showing MyHC protein expression (left panel). Quantification of MyHC+ cells (right panel). n=4, mean ± s.d., unpaired Student’s t-test, **P<0.01 (scale: 100μm, left panel). (M) QRT-PCR analysis for the indicated genes in differentiating myoblast cultures. n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01, ***P<0.001. (N) Flow cytometric quantification of OCT4-GFP+ hESCs infected with the empty retroviral vector PCLP or PCLP-KDM4B and cultured in mTeSR1 and mTeSR1 lacking bFGF and TGFβ.

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques: ChIP-sequencing, RNA Sequencing Assay, Infection, Expressing, Immunofluorescence, Quantitative RT-PCR, Plasmid Preparation, Cell Culture

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Gene tracks showing RNA-seq data. (B) Single cell RNA-seq data for DDX6 expression in human preimplantation embryos (Petropoulos et al., 2016). Epi: Epiblast; Pe: Primitive Endoderm; TE: trophectoderm. (C) RNA-seq and protein expression data for DDX6 in primed and naïve hESCs (Di Stefano et al., 2018). For RNA-seq data, n=5, mean ± s.d., unpaired Student’s t-test, ***P<0.001. For proteomic data, n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01. (D) Analysis of repetitive element expression. Repeats with significant expression differences are indicated in red (FC>1.5, FDR <0.05). (E) Differentially methylated promoter regions in DDX6 depleted cells relative to control cells. Significantly hypomethylated promoters are shown in red (>10% difference, P<0.01); significantly hypermethylated promoters are shown in blue (>10% difference, P<0.01). (F) PCA analysis of RNA-seq data for the indicated samples based on differentially expressed genes between shDDX6 #1 and shCTRL hESCs. (G) Flow cytometric detection of ΔPE OCT4-GFP+ cells after reversion of primed hESCs to a naïve state in 5i/LAF medium. Black curve shows the negative control. (H) QRT-PCR analysis for the indicated genes after 8 days of 5i/LAF treatment. Values are represented respect to control cells at day 0. n=3, mean ± s.d., unpaired Student’s t-test, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques: RNA Sequencing Assay, Expressing, Methylation, Negative Control, Quantitative RT-PCR

Journal: Cell stem cell

Article Title: The RNA helicase DDX6 controls cellular plasticity by modulating P-body homeostasis

doi: 10.1016/j.stem.2019.08.018

Figure Lengend Snippet: (A) Summary of phenotypes in DDX6 depleted stem cell populations. (B) Model proposing how DDX6 impacts cell fate through modulation of P-body homeostasis.

Article Snippet: In total, 6 g of either DDX6 antibody (Novus Biologicals) or rabbit IgG control (AbCam) were added per 100 μl of pre-cleared lysate.

Techniques:

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 1. COPII-mediated cargo transport continues in the absence of Sar1 (A) Cartoon depicting the human SAR1A and SAR1B genomic loci. The positions of the gRNAs used during CRISPR-Cas9 editing (red lines) are highlighted, and the sizes of exons (shown as green boxes) are 1/50 that of introns (shown as black lines). (B) Representative immunoblots of extracts generated from a CRISPR-modified cell line lack- ing Sar1a and subjected to siRNA-mediated treatments as shown, using antibodies directed against Sar1 and actin. Extracts were generated at the time point indicated following siRNA treat- ment. (C) Quantification of the percentage of Sar1 re- maining at different time points following Sar1b siRNA treatment (relative to mock siRNA treat- ment). Error bars represent mean ± SEM (n = 4 biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (D) Spinning disk confocal microscopy was used to image control RPE1 cells and cells lacking Sar1a either in the presence or in the absence of Sar1b, each expressing ss-DsRed following treatment with SLF (50 mM) to induce cargo disaggregation and release from the ER. Repre- sentative time-lapse images are shown (n = 15 cells, each condition; at least three biological replicates). Scale bar, 5 mm. (E and F) Quantification of cargo (E, ss-DsRed; F, ManII-SBP-GFP) accumulation within the peri- nuclear region (GM130 positive) in the various mutant backgrounds indicated. Error bars repre- sent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to the 0 min time point). See also Figures S1–S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: CRISPR, Western Blot, Generated, Confocal Microscopy, Control, Expressing, Mutagenesis

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 2. Loss of Sar1 alters the distribution of COPII coat subunits but fails to block their ability to co-assemble (A) Genome-edited cells expressing HaloTag- Sec23a and lacking Sar1a (mock transfected) and those depleted of Sar1b for 48 h were imaged live using spinning disk confocal microscopy following labeling with JFX646-HaloTag ligand. Scale bar, 5 mm; inset bar, 2 mm. (B and C) Quantification of the number of Sec23a- positive sites in cells lacking Sar1a in the presence and absence of Sar1b is shown at various time points following Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test, relative to mock treatment (B) or the 48 h time point (C). (D) Cells lacking Sar1a, depleted of Sar1b, and co- expressing HaloTag-Sec23a and YFP-Sec31a were imaged live using spinning disk confocal microscopy following labeling using JFX646- HaloTag ligand. Representative zoomed images are shown (n = 10 cells; at least three biological replicates each). Scale bar, 2 mm. (E) The volume distribution of Sec23a-positive structures in the absence of Sar1a is shown following 48 h of mock treatment or Sar1b deple- tion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to mock treatment). (F) Cells lacking Sar1a (mock transfected) or depleted of Sar1b for 72 h were immunostained using antibodies directed against Sec24a and TFG (shown only in insets). Representative confocal images (maximum intensity projections) are shown, and an arrow indicates co-localization of Sec24a and TFG. Scale bar, 5 mm; inset bar, 2 mm. (G–I) HaloTag-Sec23a in cells lacking Sar1a, either in the presence or in the absence of Sar1b, were labeled with JFX646-HaloTag ligand and sub- jected to photobleaching. Fluorescence recovery is depicted for various-sized HaloTag-Sec23a structures (n = 15 cells each; at least three bio- logical replicates each). See also Figures S4–S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Blocking Assay, Expressing, Transfection, Confocal Microscopy, Labeling, Fluorescence

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 3. COPII condensates associate with secretory cargoes that leave the ER in the absence of Sar1 (A) Cells expressing ss-DsRed and lacking both Sar1 isoforms were immunostained using antibodies directed against Sec24a and GM130 following treatment with SLF (50 mM) and imaged using STED microscopy. Representative images are shown (n = 15 cells; at least three biological replicates). Scale bar, 2 mm. (B) Cells expressing EGFP-Sec61b and HaloTag-Sec23a in the absence of Sar1 were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following cargo release and labeling with JFX646-HaloTag ligand. Violin plots show the relative displacement of each marker over time (n = 10 cells; three biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test, compared with Sec61b displacement. (C) Cells lacking both Sar1 isoforms and expressing native EGFP-Sec61b and HaloTag-Sec23a were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following treatment with SLF (50 mM) and labeling with JFX646-HaloTag ligand. Representative images are shown (n = 7 cells; three biological replicates). Arrowheads indicate accumulation of ss-DsRed with COPII condensates, which ultimately move away from their site of origin. Scale bar, 2 mm. (D) Relative fluorescence intensities of cargo (ss-DsRed) and COPII (HaloTag-Sec23a) were measured at ER subdomains over time. Error bars represent mean ± SEM. An asterisk highlights the time point at which cargo and COPII undergo a >1 mm displacement, and all measurements are aligned with respect to this time point (n = 10 cells; three biological replicates). (E) Expression of 43FM-HaloTag-L1CAM was transiently induced in control cells co-expressing Sar1 (H79G) or in cells lacking Sar1a and depleted of Sar1b, each labeled with JFX646-HaloTag ligand, and subjected to photobleaching after treatment with DDS for 60 min. Normalized fluorescence recovery in each case is shown (n = 10 cells each; at least three biological replicates each), and error bars represent mean ± SEM. An asterisk highlights the time point at which individual sites exhibiting elevated 43FM-HaloTag-L1CAM fluorescence were bleached. (F) Cells lacking Sar1a and natively co-expressing EGFP-Sec61b and HaloTag-Sec23a in the presence and absence of Sar1b were transfected with a construct encoding ss-DsRed and imaged as described for (B). The length of time ss-DsRed remained associated with HaloTag-Sec23A prior to undergoing displacement (more than 1 mm) was determined in each case. **p < 0.01, calculated using a t test, compared with cells lacking only Sar1a. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Expressing, Microscopy, Transfection, Construct, Confocal Microscopy, Labeling, Marker, Control

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 4. The absence of Sar1 destabilizes ERGIC membranes (A, B, D, E, and F) Cells lacking Sar1a (mock transfected or depleted of Sar1b for 72 h) were immunostained using antibodies directed against Sec16a (A), Tango1 (B), ERGIC-53 (and Sec16a shown in insets, with arrows highlighting their juxtaposed distribution) (D), TFG (E), or COPB1 (and GM130 shown in insets) (F). Representative confocal images (maximum intensity projections) are shown. Scale bars, 5 mm; inset bars, 2 mm. (C) Quantification of the fold change in the fluorescence intensities of Sec31a, Sec16a, and Tango1 in the absence of Sar1 (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (G) Representative immunoblots of extracts generated from control and CRISPR-modified cell lines lacking Sar1a and either in the presence or in the absence of Sar1b, using antibodies directed against COPB1 and actin. (H) Quantification of the fold change in COPB1 levels in the absence of Sar1 (relative to mock siRNA treatment of cells lacking only Sar1a). Error bar represents mean ± SEM (n = 4 biological replicates). See also Figure S8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Transfection, Western Blot, Generated, Control, CRISPR

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 5. The absence of Sar1 leads to the formation of COPII condensates that inter- fere with secretory cargo movement through the Golgi (A) Time-lapse spinning disk confocal microscopy was used to image cargo (ss-DsRed) accumulated in the perinuclear region of cells lacking Sar1 following addition of 1,6-hexanediol (top) or 2,5- hexanediol (bottom). Representative images are shown (n = 10 cells; three biological replicates). Scale bar, 5 mm. (B) Quantification of cargo (ss-DsRed) remaining within the perinuclear region of cells lacking Sar1 following 30 min of incubation with 1,6-hexanediol or 2,5-hexanediol (relative to the 0 min time point). Error bars represent mean ± SEM (n = 10 cells each; three biological replicates). ***p < 0.001, calculated using a t test, compared with treatment with 2,5-hexanediol. (C) Representative electron micrographs taken within the perinuclear region of high-pressure- frozen cells lacking Sar1a following a mock siRNA treatment or Sar1b depletion. The nuclear enve- lope (NE) is indicated in each image (n = 5 cells each; at least three biological replicates). Scale bar, 500 nm. See also Figure S9.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Confocal Microscopy, Incubation

Journal: Cell Death & Disease

Article Title: RNA decay in processing bodies is indispensable for adipogenesis

doi: 10.1038/s41419-021-03537-7

Figure Lengend Snippet: A Phase-contrast microscopy images during adipogenesis. 3T3L1 preadipocytes were differentiated into adipocytes using an adipogenic differentiation cocktail containing dexamethasone, IBMX, and insulin. On day 8, the cells were fixed and stained with oil red O. The scale bar is 100 μm. B RT-qPCR analysis of gene expression in 3T3L1 preadipocytes during adipogenesis. Individual RNA expression levels were normalized to Gapdh expression levels. The error bars indicate the SDs ( n = 3). C Immunocytochemistry images during adipogenesis. Formation of Ddx6 foci was observed. The scale bar is 50 μm. Nuclei were stained with DAPI. D Ratio of the number of cells with Ddx6 foci to the number of DAPI-positive cells each day following adipogenic induction analyzed in three sessions. Independent researchers chose 7–5 microscopic fields in a session at random. E Protein ( n = 4) and gene ( n = 6) expression analysis of Ddx6 during adipogenesis. The error bars indicate the SDs.

Article Snippet: To prepare lentiviruses for TOM and Ddx6 gene disruption, lentiCRISPRv2–sgRNA TOM and Ddx6 transfer plasmids were cotransfected with the packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260, respectively).

Techniques: Microscopy, Staining, Quantitative RT-PCR, Gene Expression, RNA Expression, Expressing, Immunocytochemistry

Journal: Cell Death & Disease

Article Title: RNA decay in processing bodies is indispensable for adipogenesis

doi: 10.1038/s41419-021-03537-7

Figure Lengend Snippet: A Western blotting of 3T3L1 Ddx6-KO preadipocytes. B Phase-contrast microscopic images during adipogenesis. On day 8, cells were fixed and stained with oil red O. The scale bar is 100 μm. C Relative absorbance of oil red O. The error bars indicate the SDs ( n = 3). Double asterisks (**) indicate significance ( P < 0.01). D Immunocytochemistry of Ddx6 foci and the proportion of cells with foci during adipogenesis. The scale bar is 50 μm. The error bars indicate the SDs ( n = 6). E Immunocytochemistry of 4E-T foci and the proportion of cells with foci during adipogenesis. The scale bar is 50 μm. The error bars indicate the SDs ( n = 6). F RT-qPCR analysis of gene expression during adipogenesis. Individual RNA expression levels were normalized to Gapdh expression levels. The error bars indicate the SDs ( n = 3).

Article Snippet: To prepare lentiviruses for TOM and Ddx6 gene disruption, lentiCRISPRv2–sgRNA TOM and Ddx6 transfer plasmids were cotransfected with the packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260, respectively).

Techniques: Western Blot, Staining, Immunocytochemistry, Quantitative RT-PCR, Gene Expression, RNA Expression, Expressing

Journal: Cell Death & Disease

Article Title: RNA decay in processing bodies is indispensable for adipogenesis

doi: 10.1038/s41419-021-03537-7

Figure Lengend Snippet: A Immunocytochemistry of 3T3L1 preadipocytes during adipogenesis. Foci of Ddx6 and 4E-T proteins were merged. The scale bar is 50 μm. Nuclei were stained with DAPI. B Immunoprecipitates bound by Ddx6 or 4E-T antibodies were analyzed by western blotting with anti-Ddx6 and anti-4E-T antibodies. C RT-qPCR analysis of 4E-T expression on each day. Individual RNA expression levels were normalized to Gapdh expression levels. The error bars indicate the SDs ( n = 3). D Phase-contrast microscopy images during adipogenesis. On day 8, the cells were fixed and stained with oil red O. The scale bar is 100 μm. E Relative absorbance of oil red O. The error bars indicate the SDs ( n = 3). Double asterisks (**) indicate signific ance ( P < 0.01). F Immunocytochemistry of 3T3L1 4E-T-KD preadipocytes during adipogenesis. The scale bar is 50 μm. The nuclei were stained with DAPI.

Article Snippet: To prepare lentiviruses for TOM and Ddx6 gene disruption, lentiCRISPRv2–sgRNA TOM and Ddx6 transfer plasmids were cotransfected with the packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260, respectively).

Techniques: Immunocytochemistry, Staining, Western Blot, Quantitative RT-PCR, Expressing, RNA Expression, Microscopy

Journal: Cell Death & Disease

Article Title: RNA decay in processing bodies is indispensable for adipogenesis

doi: 10.1038/s41419-021-03537-7

Figure Lengend Snippet: A Volcano plots at the early phase of adipogenesis in TOM-transfected and 3T3L1 Ddx6-KD preadipocytes. B Heatmap of 12 selected genes whose expression was reduced twofold in 3T3L1 TOM cells but not in 3T3L1 Ddx6-KD cells. C Venn diagram showing the number of mRNAs relevant to each segment. D Protein–protein interaction network of 12 intersecting genes predicted by STRING analysis.

Article Snippet: To prepare lentiviruses for TOM and Ddx6 gene disruption, lentiCRISPRv2–sgRNA TOM and Ddx6 transfer plasmids were cotransfected with the packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260, respectively).

Techniques: Transfection, Expressing

Journal: Molecular cell

Article Title: P-Body Purification Reveals the Condensation of Repressed mRNA Regulons.

doi: 10.1016/j.molcel.2017.09.003

Figure Lengend Snippet: Figure 3. The P-Body Proteome Does Not Contain Any Ribosomal Subunits and Forms an Interaction Network Distinct from SGs (A) In situ hybridization combined with immuno-electron microscopy. The 18S and 28S rRNAs (10 nm gold particles) were excluded from DDX6 immuno-labeled (15 nm gold particles) P-bodies (dashed lined). Scale bars, 200 nm. Average densities ± SD of 18S and 28S probes were quantified in P-bodies, their immediate vicinity, and the surrounding cytosol. Quantifications in arsenite-induced SGs are shown for comparison. (B) The Venn diagram shows limited overlap between P-body proteome and previously reported SG proteome (upper left panel). Specific protein interactions segregate P-body proteins from SG ones (right panel), and create a denser network in P-bodies than in SGs (lower left panel). (C) RNA-binding proteins were more enriched in sorted P-bodies than in purified SGs. Domain homology analysis further revealed that RNA-binding proteins represent up to 70% of the P-body proteome.

Article Snippet: Primary antibodies were goat 4E-T and rabbit EDC3, PUM1, PUM2 (Abcam), rabbit DDX6 (Novus), goat TIA1, IGF2BP1, IGF2BP3 and mouse EDC4 (Santa Cruz Biotechnology), rabbit LSM14A (Merck-Millipore).

Techniques: In Situ Hybridization, Immuno-Electron Microscopy, Labeling, Comparison, RNA Binding Assay

Journal: bioRxiv

Article Title: GC content shapes mRNA decay and storage in human cells

doi: 10.1101/373498

Figure Lengend Snippet: (A) mRNA stabilization after DDX6 silencing in HEK293 and K562 cells applies to GC-rich mRNAs. The fold-changes (FC) in mRNA accumulation (in green) were analyzed as in . (B) mRNA translation derepression after DDX6 silencing in HEK293 cells applies to AU-rich mRNAs. The fold-changes in translation rate (in orange) were analyzed as in (A). (C) GC-rich mRNAs are particularly enriched in the DDX6 CLIP experiment (in dark green). See also Figures S3-5.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-DDX6 (1:15000; Novus Biological), rabbit polyclonal anti-ribosomal S6 (1:5000; Cell Signaling Technology), anti-XRN1 (1:1000, Bethyl Laboratories), anti-XRN1 (1:5000 Novus Bioscience), anti-Pol II (1:100, Santa Cruz).

Techniques:

Journal: bioRxiv

Article Title: GC content shapes mRNA decay and storage in human cells

doi: 10.1101/373498

Figure Lengend Snippet: (A) mRNA stabilization after XRN1 silencing in HeLa and HCT116 cells (in brown) applies to GC-rich mRNAs. The analysis was performed as in . The GC content distribution for all mRNAs is presented for comparison (in grey). (B) mRNA stabilization after PAT1B silencing in HEK293 cells (in peach) applies to AU-rich mRNAs. The analysis was performed as in (A). (C) Read coverage of XRN1 targets (FC>1, n=333) and non-targets (FC<-1, n=139), as defined in the siXRN1 dataset. Their average read coverage was analyzed in control cells (upper panel) and after XRN1 silencing (lower panel), and normalized as described in the Methods. (D) Read coverage of PAT1B targets (FC>0.6, n=616) and non-targets (FC<-0.6, n=493), as defined in the siPAT1B dataset. The data were analyzed as in (C). (E,F) Clustering analysis of mRNAs depending on their GC content, their differential expression after silencing DDX6, XRN1 or PAT1B, and their enrichment in PBs. Raw GC content and log2 transformed ratio of the other datasets were used for the clustering of both transcripts (lines) and datasets (columns). The values were color-coded as indicated on the right scale, and the Spearman correlation matrix is presented in F (all p-values <10-48). The heatmap highlights the distinct fate of GC-rich and AU-rich mRNAs. See also Figures S4 and S6.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-DDX6 (1:15000; Novus Biological), rabbit polyclonal anti-ribosomal S6 (1:5000; Cell Signaling Technology), anti-XRN1 (1:1000, Bethyl Laboratories), anti-XRN1 (1:5000 Novus Bioscience), anti-Pol II (1:100, Santa Cruz).

Techniques: Comparison, Expressing, Transformation Assay