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. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, unpaired Student’s t test. n=3, mean ± s.d. See also Figure S5 and Tables S1 and S2.

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: Protein-Protein interactions, 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, Control, 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). See also Figures S1, S2 and 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: Quantitative RT-PCR, Immunofluorescence, Expressing, RNA Sequencing

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. See also Figure S7 and Table S4.

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, Infection, Control, Expressing, Immunofluorescence, Quantitative RT-PCR, Retroviral, 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. See also Figure S4.

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, Expressing, Methylation, Control, 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: Nature Communications

Article Title: HNRNPK maintains epidermal progenitor function through transcription of proliferation genes and degrading differentiation promoting mRNAs

doi: 10.1038/s41467-019-12238-x

Figure Lengend Snippet: HNRNPK is necessary for DDX6 to bind differentiation associated mRNAs. a RNA IP was performed in CTLi and HNRNPKi cells using a DDX6 antibody. RT-QPCR was used to determine the levels of binding between DDX6 and differentiation associated mRNAs in the presence or absence of HNRNPK. IGG IPs in CTLi and HNRNPKi cells were used as specificity controls. Binding was calculated as a percent of input. b RNA IP was performed in CTLi and DDX6i cells using a HNRNPK antibody. RT-QPCR was used to determine the levels of binding between HNRNPK and differentiation associated mRNAs in the presence or absence of DDX6. IGG IPs in CTLi and DDX6i cells were used as specificity controls. n = 4. c Western blot analysis of HNRNPK and DDX6 protein levels upon HNRNPK or DDX6 knockdown. d Immunoprecipitations (IPs) were performed using either an HNRNPK or DDX6 antibody or IGG and Western blotted for HNRNPK or DDX6 protein expression. IPs were performed +/− RNase A. Five percent of the cell lysate was used as input. Representative blots are shown. n = 3 independent experiments performed for Fig. 3 unless otherwise indicated. All error bars = SD. **** p < 0.0001, *** p < 0.001 (2 way ANOVA followed by Tukey’s multiple comparison test for a , b )

Article Snippet: Three microgram of HNRNPK antibody (Bethyl Laboratories: A300–674A), DDX6 (Novus Biologicals: NB200–192), RNA Pol II (Active motif: 39097), CDK9 (Bethyl Laboratories: A303–493A) or rabbit IgG were complexed with 50 μl of Protein G Dynabeads (Life Technologies: 10004D) at room temperature for 30 min The antibody conjugated Dynabeads were then incubated with cell lysates on a rotator at 4 °C overnight.

Techniques: Quantitative RT-PCR, Binding Assay, Western Blot, Knockdown, Expressing, Comparison

Journal: Nature Communications

Article Title: HNRNPK maintains epidermal progenitor function through transcription of proliferation genes and degrading differentiation promoting mRNAs

doi: 10.1038/s41467-019-12238-x

Figure Lengend Snippet: HNRNPK binding to proliferation genes is not dependent on active transcription and model of HNRNPK regulation of epidermal stem and progenitor cell self-renewal through mRNA transcription and degradation. a – c Keratinocytes were treated +/− actinomycin D (ACTD) to inhibit transcription. ChIP was performed using a HNRNPK or RNA Pol II CTD phospho Ser2 antibody in +/− actinomycin D (ACTD) treated cells. ChIP was also performed using IGG in +/− ACTD treated cells as a specificity control. Primers for each gene was designed at the 5’ end (genomic region #1) and past the 3’ end (genomic region #2) of each proliferation gene ( MYC , EGFR , and CYR61 ). Binding to each gene was calculated as a percentage of input. Mean values are shown with error bars = SD, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test ( a – c ). n = 2. d Top panel: HNRNPK recruits DDX6 to degrade mRNAs that code for potent differentiation promoting transcription factors to prevent premature differentiation of epidermal cells. Bottom panel: HNRNPK recruits RNA polymerase II to self-renewal and proliferation genes to promote epidermal self-renewal

Article Snippet: Three microgram of HNRNPK antibody (Bethyl Laboratories: A300–674A), DDX6 (Novus Biologicals: NB200–192), RNA Pol II (Active motif: 39097), CDK9 (Bethyl Laboratories: A303–493A) or rabbit IgG were complexed with 50 μl of Protein G Dynabeads (Life Technologies: 10004D) at room temperature for 30 min The antibody conjugated Dynabeads were then incubated with cell lysates on a rotator at 4 °C overnight.

Techniques: Binding Assay, Control, Comparison

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Purification of DDX6 complexes by TAP-tag. (A) HEK293 cells were transfected with a FLAG-DDX6-HA or an empty plasmid as a control. After 48 h, proteins were analyzed by Western blotting with anti-DDX6 antibodies. (B) In parallel, transfected cells were stained with anti-FLAG (red) and anti-EDC4 (green) antibodies to detect the exogenous DDX6 protein and P-bodies, respectively. For comparison, untransfected cells were stained with anti-DDX6 and anti-EDC4 antibodies. Owing to the round shape of the cells, the maximal projection of a z-series of images is shown to better visualize P-bodies. Scale bar: 10 μm. (C) DDX6 enrichment in P-bodies with respect to the surrounding cytoplasm ( n = 94 and 90 for transfected and untransfected samples, respectively) was quantified from single-plane images. (D) Endogenous DDX6 was detected in untransfected HEK293 cells by immunoelectron microscopy, as previously described ( Souquere et al. , 2009 ). One P-body highly enriched in DDX6 is shown. (E) Cells transfected with a FLAG-DDX6-HA or an empty plasmid were lysed in the presence of RNase inhibitor or RNase, and DDX6 complexes were purified by TAP-tag. Proteins were migrated on a denaturing gel along with a molecular weight marker (MW) and silver stained.

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Purification, Transfection, Plasmid Preparation, Control, Western Blot, Staining, Comparison, Immuno-Electron Microscopy, Molecular Weight, Marker

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Top DDX6 partners.

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Functional classification of DDX6 partners.

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Functional Assay, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Characterization of decay and repression complexes in untransfected cells. (A and B) HEK293 cytoplasmic lysates were immunoprecipitated with anti-DDX6 antibodies and analyzed by Western blotting for components of (A) decapping and (B) repression complexes, as indicated. For DDX6 and EDC3, only one-tenth of the immunoprecipitate was loaded on the gel to avoid saturation. For facilitation of comparison, the immunoprecipitation efficiency was estimated from scanned images, as described in Materials and Methods , and indicated below as percent values. (C) Cytoplasmic lysates from HEK293 incubated or not with cycloheximide (+ CHX) were separated by centrifugation on sucrose gradients and analyzed by optical densitometry (top panels). Proteins from collected fractions were analyzed by Western blotting with anti-DDX6 antibodies (bottom panels).

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Immunoprecipitation, Western Blot, Comparison, Incubation, Centrifugation

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Characterization of the ATXN2/2L complex and EJC in untransfected cells. (A) Components of the ATXN2/2L complex were analyzed as in . (B) HeLa cells were transfected with indicated siRNAs. After 48 h, cells were stressed with arsenite for 30 min. Localization of ATXN2, ATXN2L, and DDX6 in stress granules was analyzed by immunofluorescence, using TIA1 as a stress granule marker. Scale bar: 10 μm. (C) Components of the EJC were analyzed as in A.

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Transfection, Immunofluorescence, Marker

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Role of P-body proteins in the maintenance of P-bodies. (A–C) HeLa cells were transfected with indicated siRNAs. After 48 h, (A) proteins were analyzed by Western blotting with indicated antibodies, and (B) P-bodies were analyzed by immunofluorescence with anti-EDC4 or DDX6 antibodies, along with antibodies directed against the silenced protein to check the silencing at the individual cell level (unpublished data). Scale bar: 10 μm. (C) P-bodies were counted in three independent experiments, and their average number per cell was plotted. **, p < 0.005. (D and E) After transfection of siLSM14A and siLSM14B, proteins were analyzed by Western blotting using indicated antibodies (D). Signals were quantified in three independent experiments and plotted (E).

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Transfection, Western Blot, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes

doi: 10.1091/mbc.E15-03-0136

Figure Lengend Snippet: Role of P-body proteins in P-body assembly after arsenite treatment. HeLa cells were transfected with indicated siRNAs and analyzed as in , except that cells were treated with arsenite for 30 min before fixation. P-bodies were immunostained (A) and counted (B) as in (all), except that LSM14A antibodies were used in place of DDX6, as this accumulated in both stress granules and P-bodies. A second quantitation restricted to P-bodies larger than 450 nm was performed to show the characteristic defect observed after silencing LSM14A (large). Scale bar: 10 μm. The corresponding Western blot analysis is presented in Supplemental Figure 2D.

Article Snippet: Primary antibodies included mouse ATXN2 and G3BP from BD Biosciences; rabbit ATXN2L from Bethyl; goat TIA1 and mouse EDC4, eIF4E, and XRN1 from Santa Cruz Biotechnology (Heidelberg, Germany); goat 4E-T and rabbit LSM12 from Abcam (Paris, France); rabbit LSM14A and mouse PABPC1 from Merck-Millipore (Molsheim, France); rabbit LSM14B and mouse α-tubulin from Sigma-Aldrich; rabbit DDX6 from Novus Biologicals (Bio Techne, Lille, France); and rabbit ribosomal S6 from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelynes, France).

Techniques: Transfection, Quantitation Assay, Western Blot