Journal: Communications Biology

Article Title: Single-nucleus RNA sequencing and functional studies of acute methamphetamine-induced cognitive impairment

doi: 10.1038/s42003-026-09728-2

Figure Lengend Snippet: A UMAP plot showing the expression of Ddx5 between Meth and control groups. B , C Ddx5 mRNA and protein levels in hippocampus ( n = 3). D Hippocampus stained with Ddx5 antibody and quantification of the relative fluorescence intensity ( n = 8), scale bar = 100 μm. E IHC staining of Ddx5 in hippocampus, scale bar = 20 μm. F, G Ddx5 mRNA and protein levels in primary neurons ( n = 3). H Primary neurons stained with Ddx5 antibody and quantification of the relative fluorescence intensity ( n = 6), scale bar = 5 μm. I Dendrogram visualizing the hierarchical structure of the co-expression network, where each module color represents a distinct module. J The top 20 hub genes identified on kME. K Radar plot showing the expression of modules in major hippocampal cell types. L Heatmap showing the correlation between the five modules and cognitive function. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. Control group.

Article Snippet: The primary antibodies used were: PEX5 (12545-1-AP, 1:1000) and Ddx5 (10804-1-AP, 1:1000) from Proteintech; β-actin (sc-47778, 1:500) from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Staining, Fluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Regulation of Sox8 through lncRNA Mrhl mediated chromatin looping in mouse spermatogonia

doi: 10.1101/2021.10.05.463295

Figure Lengend Snippet: (A) The mouse Sox8 locus on chromosome 17 where Mrhl lncRNA binds at the bidirectional promoter 140bp upstream of the TSS in a p68-dependent manner to maintain Sox8 in the transcriptionally repressed state. (B) EMSA performed for the indicated DNA oligo incubated with RNA oligo TFO1. The lanes have the reaction components as indicated above them (C) EMSA performed with mutant TTS2 and RNA TFO1 where no shift in mobility is observed (D) EMSA for negative control TFO/TTS pair (E) Artificial spectrum generated by summation of individual CD spectra recorded for TTS2 only and NC TFO only (red) overlaid with CD spectrum of triplex reaction for the oligonucleotides, (F) Artificial spectrum generated by summation of individual CD spectra recorded for TTS2 only and TFO1 only (red) overlaid with CD spectrum of triplex reaction for the same oligonucleotides. Plots are an average of 4 independently recorded spectra. (G) Results of the in-nucleus triplex pulldown assay show significant enrichment of the Sox8 ITS region in the TFO1 oligo associated chromatin fraction over NC TFO associated chromatin fraction both without and with RNaseH digestion. Data in the graph has been plotted as mean ±S.D., N=3. *** P ≤ 0.0005, ** P ≤ 0.005, * P ≤ 0.05, N.S - Not significant (two-tailed Student’s t test)

Article Snippet: [γ-32P]ATP was sourced from List of antibodies that were used are as follows with the manufacturer and catalog number in parenthesis are: CTCF (AbCam; ab 188408), (Cell Signaling Technology; 3418), Rad21 (AbCam; ad9920), p68/DDX5 (Cusabio; CSB-PA003685), YY1 (Diagenode; C15410345), H3K4mel (Diagenode; C15410037), H3K27ac (Diagenode; C15410174), Ezh2 (Diagenode; C15410039).

Techniques: Incubation, Mutagenesis, Negative Control, Generated, Circular Dichroism, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of Sox8 through lncRNA Mrhl mediated chromatin looping in mouse spermatogonia

doi: 10.1101/2021.10.05.463295

Figure Lengend Snippet: (A) Sox8 locus harbouring CTCF binding site within exon 3. (B) Mrhl expression levels in mESC and adult brain cortex (C) Sox8 expression in mESC and adult brain cortex (D) Analysis of ChIP-seq datasets in mESC showing the presence of CTCF and cohesin (SMC1) at both exon 3 and the promoter (E) Analysis of ChIP-seq datasets in adult brain cortex showing reduced occupancy of CTCF and cohesin (SMC1) at exon 3 and promoter. Results for ChIP-qPCR for (F) CTCF, (G) Rad21 and (H) p68 showing their occupancy patterns at the promoter and exon3 of Sox8 gene in cells without and with Wnt activation. Data in the graph has been plotted as mean ±S.D., N=3. *** P ≤ 0.0005, ** P ≤ 0.005, * P ≤ 0.05, N.S - Not significant (two-tailed Student’s t test)

Article Snippet: [γ-32P]ATP was sourced from List of antibodies that were used are as follows with the manufacturer and catalog number in parenthesis are: CTCF (AbCam; ab 188408), (Cell Signaling Technology; 3418), Rad21 (AbCam; ad9920), p68/DDX5 (Cusabio; CSB-PA003685), YY1 (Diagenode; C15410345), H3K4mel (Diagenode; C15410037), H3K27ac (Diagenode; C15410174), Ezh2 (Diagenode; C15410039).

Techniques: Binding Assay, Expressing, ChIP-sequencing, ChIP-qPCR, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of Sox8 through lncRNA Mrhl mediated chromatin looping in mouse spermatogonia

doi: 10.1101/2021.10.05.463295

Figure Lengend Snippet: (A) Cohesin subunit SMC3 and Rad21 are found to bind close to the CTCF binding site in exon 3 of Sox8 (B) CTCF appears to be bound at the binding site within Sox8 in those tissue in which mrhl is expressed but not in some other in which mrhl isn’t expressed (C) ChIP WB for CTCF in Gcl-spg cells with and without Wnt induction. (D) ChIP WB for Rad21 in Gcl-spg cells with and without Wnt induction (E) ChIP WB for p68 in Gcl-spg cells with and without Wnt induction (F) ChIP WB for CTCF in mice testes of ages 7 days and 21 days (G) ChIP WB for Rad21 in mice testes of ages 7 days and 21 days (H) ChIP WB for p68 in mice testes of ages 7 days and 21 days

Article Snippet: [γ-32P]ATP was sourced from List of antibodies that were used are as follows with the manufacturer and catalog number in parenthesis are: CTCF (AbCam; ab 188408), (Cell Signaling Technology; 3418), Rad21 (AbCam; ad9920), p68/DDX5 (Cusabio; CSB-PA003685), YY1 (Diagenode; C15410345), H3K4mel (Diagenode; C15410037), H3K27ac (Diagenode; C15410174), Ezh2 (Diagenode; C15410039).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Regulation of Sox8 through lncRNA Mrhl mediated chromatin looping in mouse spermatogonia

doi: 10.1101/2021.10.05.463295

Figure Lengend Snippet: (A) The two different regions within Mrhl targeted by the two shRNA (B) Mrhl silencing efficiency of the two shRNA-silencing efficiencies of ∼65% and ∼55% respectively were observed for Mrhl while the transcript levels of the host phkb gene were not perturbed significantly. Results for ChIP-qPCR for (C) CTCF, (D) Rad21 of cohesin and (E) p68 show significant reduction in occupancy of CTCF at both the promoter and exon 3 of Sox8 upon induction of silencing of Mrhl with both shRNA construct 1 and shRNA construct 2. Occupancy of (F) CTCF (G) Rad21 and (H) p68 is observed at the promoter and exon 3 of Sox8 locus in P7 mice testes and a significantly reduced occupancy is observed in P21 mice testes. Data in the graph has been plotted as mean ± S.D., N=3. *** P ≤ 0.0005, ** P ≤ 0.005, * P ≤ 0.05, N.S - Not significant (two-tailed Student’s t test)

Article Snippet: [γ-32P]ATP was sourced from List of antibodies that were used are as follows with the manufacturer and catalog number in parenthesis are: CTCF (AbCam; ab 188408), (Cell Signaling Technology; 3418), Rad21 (AbCam; ad9920), p68/DDX5 (Cusabio; CSB-PA003685), YY1 (Diagenode; C15410345), H3K4mel (Diagenode; C15410037), H3K27ac (Diagenode; C15410174), Ezh2 (Diagenode; C15410039).

Techniques: shRNA, ChIP-qPCR, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) The presence and location of the predicted prion-like (PrD) domain in the DDX5 protein is conserved between killifish and humans. Red line: PLAAC score of the protein sequence. DDX5 amino acid positions are indicated at the bottom. Amino-acid composition is color-coded for visualization. (B) The presence and location of the predicted intrinsically disordered region (IDR) in the DDX5 protein is conserved between killifish and humans. Blue line: predicted amino acid disorder score based on DISOPRED3. (C) Immunohistochemistry for DDX5 in brain sections from young (3.5 months) and old (7 months) male killifish. Green: DDX5, Blue: DAPI (nuclei). Image representative of 3 individual fish for each age group. Staining was performed twice independently, with a total of ∼7 sections per fish per experiment. Scale bar: 10µm. Quantification of the subcellular localization of DDX5 puncta is in . (D) Immunohistochemistry for DDX5 in brain sections from young (4 months) and old (28 months) male mice. Green: DDX5, Blue: DAPI (nuclei). Image representative of 2 mice per age group, with sections per mouse. Scale bar: 20µm. (E) The Tg(ddx5:DDX5-GFP) transgenic line was generated by using 5kb surrounding the transcriptional start site of the DDX5 gene (including the first exon) to drive the expression of DDX5-GFP fusion protein. As Tg(ddx5:DDX5-GFP) were not viable as F1 fish, we used F0 chimeric adult fish (∼6-7 months old) for the staining. (F) Immunohistochemistry for GFP in brain sections from old (∼6-7 months) male Tg(ddx5:DDX5-GFP) transgenic killifish. Green: GFP, Blue: DAPI (nuclei). Image representative of 3 individual fish, ∼6 sections per fish. Scale bar: 10µm. (G) Generation of killifish DDX5 mutants with truncation of the prion-like domain (DDX5-ΔPrD) or truncation of the intrinsically disordered region (DDX5-ΔIDR) tagged with EGFP for expression in mammalian cells. FL: full length. Red line: PLAAC score of the protein sequence. Bottom: Color-coded distribution of the disordered region (DISOPRED3, blue), net charge (sliding window of 5 amino acids), and hydropathy (sliding window of 5 amino acids) in the killifish DDX5 protein. (H) Percentage of aggregate-like fluorescent puncta of DDX5-EGFP full length (FL) and truncation mutants in human 293T cells. Images are representative of two independent experiments, each performed in triplicate wells. Scale bar: 10µm. The percentage of cells with DDX5-GFP aggregates (estimated based on >3 fields of view for each replicate) is indicated in the top left corner.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Sequencing, Immunohistochemistry, Staining, Transgenic Assay, Generated, Expressing

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) Location of the predicted prion-like (PrD) domain in DEAD-box helicase family members identified in this study. Red line: score of the PrD domain. Amino-acid numbers are indicated at the bottom. PrD score (PLAAC) is indicated (bottom right). Amino-acid composition is color-coded. (B) Amino acid composition of PrD and RGG (motifs rich in arginines and glycines) domains in the primary DDX5 paralog in this study, DDX5 (2 of 2). The DDX5 (1 of 2) is more prevalent in the liver. Amino-acid numbers of each domain are indicated. (C) Production and validation of killifish DDX5 C-terminus antibody using a Western blot for brain lysates, extracted from either young (∼3 month old) or old (∼7 month old) killifish. The peptide sequence used as an antigen for immunization is indicated at the bottom. (D) Immunohistochemistry of killifish brain sections using C-term DDX5 antibody (green) and phalloidin, that recognizes staining actin filaments (red). Blue: DAPI. Scale bar: 10µm. (E) Qualitative quantification of the localization of DDX5 puncta in brain sections from young (3.5 months old) and old (7 months old) killifish. Data from 3 animals for each condition, with a total of 43 sections.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Biomarker Discovery, Western Blot, Sequencing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) The Tg(ddx5:DDX5-GFP) transgenic line was generated by using 5kb surrounding the transcriptional start site of the DDX5 gene (including the first exon) to drive the expression of DDX5-GFP fusion protein (dashed blue line). Exons are indicated in red. (B) Generation of DDX5 CRISPR killifish mutant, including (1) gRNA target design for exons 5 and 7, and (2) evidence for successful germline transmission in F1 embryos. (3) F1 fries were never detected, even as heterozygous. Therefore, no stable line was generated. (C) Localization of DDX5-GFP (left) or polyQ(97)-GFP puncta in 293T cells. DDX5 puncta are primarily nuclear, whereas polyQ(97) puncta are primarily perinuclear (white arrowheads). Green: GFP-tag of the indicated protein; Blue: Hoechst. Images are representative of 2 experiments, each performed in triplicates. Scale bar: 5µm.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Transgenic Assay, Generated, Expressing, CRISPR, Mutagenesis, Transmission Assay

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) ‘One-color’ experimental design to assess protein aggregation in yeast, by conditionally expressing C-terminally EGFP-tagged killifish protein candidates under a galactose-inducible promoter. Protein aggregation is scored by quantification of the EGFP fluorescent puncta in yeast. (B-C) Identification of killifish candidate proteins that do not aggregate (B) and candidate proteins that do aggregate (C) following galactose-inducible overexpression in yeast. White arrows: fluorescent puncta (aggregates). Scale bar: 5µm. (D) ‘Two-color’ experimental design to assess prion-like aggregate propagation in yeast. This design tests whether a brief overexpression (‘induction’) of a killifish candidate (C-terminally tagged with mRuby3 and under the control of a galactose-inducible promoter) can induce a cross-generational prion-like propagation of a constitutively expressed candidate (C-terminally tagged with eGFP and under the control of the GPD-promoter with single-copy number plasmid). The cross-generational effect was tested during ‘outgrowth’ (i.e., 200-fold dilution), highlighting aggregates that can stably propagate for 7-8 generations of yeast cells. Protein aggregation is scored by quantification of mRuby (red) or EGFP (green) fluorescent puncta in yeast. (E) Using the two-color system, killifish DDX5-EGFP aggregates were visible in both induction and outgrowth phases, indicating prion-like propagation. Representative of 4 independent experiments (quantified in F). White arrows: fluorescent puncta (aggregates). Scale bar: 5µm. (F) Quantification of DDX5 aggregates and prion-like seeding potential by calculating the fraction of EGFP positive cells with EGFP puncta during pre-induction, induction, and outgrowth. Mean values from 4-6 independent experiments are indicated by the black bar (total of 6 independent pre-induction measurements and four independent experiments with matched pre-induction, induction, and outgrowth measurements). Each dot represents the average value within each experiment (an average of 135 GFP-positive yeast cells were quantified for each strain under each experimental condition). Difference across groups were assessed using Student’s t-test. (G) The prion-like domain of DDX5 is necessary for propagation. Following overexpression of DDX5FL-mRuby3, DDX5ΔPrD-GFP forms aggregates (white arrows) in the induction phase but becomes diffuse (nuclear) in the outgrowth phase. Representative of 4 independent experiments (quantified in H). Scale bar: 5µm. (H) Quantification of DDX5 ΔPrD aggregation and prion-like seeding potential by calculating the fraction of EGFP positive cells with EGFP puncta during pre-induction, induction with DDX5 FL-mRuby3, and outgrowth. The black bar indicates mean values from 4-5 independent experiments (total of 5 independent pre-induction measurements and 4 independent experiments with matched pre-induction, induction, and outgrowth measurements). Each dot represents the average value within each experiment (an average of 135 GFP-positive yeast cells were quantified for each strain under each experimental condition). Difference across groups were assessed using Student’s t-test.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Expressing, Over Expression, Control, Plasmid Preparation, Stable Transfection

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A, B) Identification of killifish candidate proteins that do not aggregate (A), and proteins that aggregate under basal expression levels (B). (C) Following overexpression of DDX5ΔPrD-mRuby3, DDX5ΔPrD-GFP form aggregates (white arrows), which become diffuse and nuclear-localized after propagation. Representative of 4 independent experiments (quantified in D). Scale bar: 5µm. (D) Quantification of DDX5 aggregates and prion-like seeding potential by calculating the fraction of GFP positive cells with EGFP puncta in the indicated systems during pre-induction, induction, and outgrowth. Mean values from 3 or more independent experiments are indicated by the black bar, and each dot represents the average value within each experiment (induction and outgrowth phase results were selected when overexpression of mRuby3-tagged protein was robust. An average of 135 GFP-positive yeast cells are quantified for each strain under each experimental condition). P-values: from unpaired t-test between indicated experimental phases.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Expressing, Over Expression

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) Left: following expression of mRuby3 alone, killifish DDX5-EGFP remains diffuse (and nuclear-localized). Representative images of 4 independent experiments (quantified on the right). Scale bar = 5 µm. Right: Quantification of DDX5 aggregation and prion-like seeding potential by calculating the fraction of EGFP positive cells with EGFP puncta during pre-induction, induction, and outgrowth. Mean values from 4 independent experiments are indicated by the black bar. Each dot represents the average value within each experiment (an average of 135 GFP-positive yeast cells were quantified for each strain under each experimental condition). Difference across groups were assessed using Student’s t-test. (B) Following expression of the toxic and aggregation-prone PolyQ(103) repeat, killifish DDX5-EGFP remains diffuse (and nuclear-localized). White arrows: PolyQ(103)-mRuby3 aggregates. Mean values from 3 independent experiments are indicated by the black bar. Each dot represents the average value within each experiment (an average of 135 GFP-positive yeast cells were quantified for each strain under each experimental condition). Difference across groups were assessed using Student’s t-test. Scale bar: 5 µm. (C) DDX5 aggregation displays species barrier. Following overexpression of human DDX5-mRuby3, killifish DDX5-GFP remains diffuse (and nuclear-localized), highlighting species barrier – another characteristic of prion-like propagation. White arrows: DDX5 (human)-mRuby3 aggregates. Representative of 3 independent experiments. Scale bar: 5 µm. (D) Fluorescence recovery after photobleaching (FRAP) experiment to compare the recovery of killifish DDX5 (blue line) and yeast Sup35NM (a bona fide yeast prion domain, red line) puncta in yeast. Left panel: Prion-like proteins exhibit little fluorescence recovery by FRAP, indicating their aggregate nature. Right panel: Representative of 2 independent experiments. The trace represents mean +/- SEM of the normalized intensity of the photobleached area at each time point (10s interval with photobleaching performed at the 4 th frame).

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Expressing, Over Expression, Fluorescence

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) Purified recombinant killifish DDX5 FL and mutants (ΔPrD and ΔIDR), as seen by Native-PAGE stained with coomassie blue (left), SDS-PAGE stained with coomassie blue (center), and SDS-PAGE blotted against DyLight 549 (SNAP-tagged protein was labeled with DyLight 549 dye prior to gel electrophoresis) (right).

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Purification, Recombinant, Clear Native PAGE, Staining, SDS Page, Labeling, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) DDX5 can phase separate as a function of protein and NaCl concentration, and its prion-like domain (PrD) and intrinsically disordered region (IDR) are important to drive this behavior. Phase separation diagrams for purified killifish DDX5 recombinant protein full length (FL) and truncation mutants (ΔPrD and ΔIDR) under variable NaCl (y-axis, NaCl in mM) and protein concentrations (x-axis, in μM, range of protein concentration was estimated from mass spectrometry data). Empty squares: Diffuse state; full circle: Condensate state. (B) Left: a DIC representative image of DDX condensates. Right: temporal dynamics of the phase separation of DDX5 protein at various temperatures for DDX5 full length (FL) or different truncation mutants (ΔPrD and ΔIDR). Protein and salt concentrations are indicated. Red: DyLight 549 labeled C-terminally SNAP-tagged recombinant protein. Representative of 2 independent experiments. Experiments in (B) were performed with 5 μM protein at 150 mM NaCl. Scale bar: 5 µm unless indicated.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Concentration Assay, Purification, Recombinant, Protein Concentration, Mass Spectrometry, Labeling

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) Experimental scheme used to measure the ATPase activity of the DDX5 in diffuse or condensates forms. Empty squares: diffuse state; full circle: condensates. Bottom panel: western blot to verify DDX5 protein concentration. (B) Assessment of ATPase activity for the killifish DDX5 protein in diffuse (red circles) or condensate (blue circles, above 2 μM) forms. ATPase activity is measured as a function of free phosphate (Pi) release rate (μM/min) at the indicated DDX5 protein concentrations (µM). Values of 4 replicates from one representative experiment are shown (corresponding to the western blot in (A)). Two independent experiments were performed and the result of the second is in . (C) Formation of less spherical (more disorganized) DDX5 aggregates over time. SNAP-tagged recombinant DDX5 was labeled with the DyLight 549 dye to allow visualization in the red channel (left panels). Quantification of the shape factor (corresponding to the sphericality of the condensate/aggregates) (right panel). P-values from a Student’s t-test. (D) ATPase activity experiment for the killifish DDX5 protein, comparing the activities of the aggregate (red or blue bars), diffuse forms (green bar), and buffer only (purple bar). ATPase activity is measured as a function of free Pi production rate (μM/min) per μM of protein. Representative of 2 many independent experiments (mean +/- SD from one experiment was shown). (E) Potential of DDX5 to form aggregates upon seeding with aggregated DDX5 protein (seed) at the indicated molar ratios (top) and time post-seeding (left). SNAP-tagged recombinant DDX5 was labeled with DyLight 549 dye to allow visualization in the red channel. The scale bar is either 25µm (top) or 50µm (bottom). Representative of 2 independent experiments.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Activity Assay, Western Blot, Protein Concentration, Recombinant, Labeling

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: (A) ATPase assay results from an additional independent experiment (similar to ). Killifish DDX5 protein in diffuse (red circles) or condensate (blue circles, above 2 μM) forms were used to catalyze ATP hydrolysis in the presence of yeast total RNA. The ATPase activity is measured as a function of free phosphate (Pi) release rate (μM/min), controlling for the same total DDX5 protein concentrations (µM). Values of 4 technical replicates are shown along with western blot to confirm a similar level of total DDX5 protein. (B) Killifish DDX5ΔPrD shows concentrations dependent ATPase activity in the presence of RNA (red circles). At these consecrations, the DDX5ΔPrD protein was in its diffuse form. ATPase activity is measured as a function of free phosphate (Pi) production rate (μM/min) at the indicated protein concentrations (µM). Values of 4 replicates from one representative experiment are shown along with SDS-PAGE of the reaction mixture. Two independent experiments were performed. (C) Left: ATPase activity for the killifish DDX5 full length (FL) and mutants (ΔPrD and ΔIDR). As negative controls, BSA (Bovine Serum Albumin) or buffer alone are used. Activity is measured under diffuse conditions (0.5 μM protein, 150 mM NaCl) as a function of free Pi production rate (μM/min) at the indicated RNA concentrations (ng/μL). Right: Inset displays activity at lower concentration with higher resolution. Mean +/- SEM from 3 independent experiments (3-4 replicates in each experiment) were shown. (D) Michaelis-Menten fit for the activity of DDX5 full length (FL) and mutants (ΔPrD and ΔIDR), as measured in (C). The Michaelis–Menten equation describes the rate of ATP hydrolysis by relating the rate of free Pi production (μM/min) catalyzed by DDX5 to the concentration of the RNA substrate (yeast total RNA) (ng/μL). The estimated maximum reaction rate (Vmax) and the Michaelis constant KM (a measure of the substrate-binding affinity) are provided.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: ATPase Assay, Activity Assay, Western Blot, SDS Page, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Identification of protein aggregates in the aging vertebrate brain with prion-like and phase separation properties

doi: 10.1101/2022.02.26.482115

Figure Lengend Snippet: Putative model for the maturation of DDX5 condensate and the associated changes in activity Putative model depicting the maturation of DDX5 condensates (active) into aggregates (inactive) with prion-like seeding potential over time or during aging.

Article Snippet: Primary antibodies used were the following: DDX5 (Atlas Antibodies, HPA020043, [1:200]) and NeuN (Millipore, MAB377, clone A60 [1:500]).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: SNHG15 Mediates MTSS1 Gene Expression via Interacting with the Gene Promoter and Regulating Transcription Pausing

doi: 10.3390/ijms252111565

Figure Lengend Snippet: DDX5 and RTF1 are two proteins associated with SNHG15. ( A ) DNA plasmids expressing full-length SNHG15 and three truncated SNHG15 samples were constructed and stably transfected into BT-549 cells. The relative position of the truncated SNHG15 is shown. ( B , C ) qRT-PCR was performed to measure the relative levels of full and truncated SNHG15 and corresponding expression of MTSS1 mRNA in individual stable cells. ** p < 0.01. * p < 0.05. ( D ) SNHG15 pulldown and MS spectrometry assays were performed to identify proteins associated with SNHG15. The table lists five proteins identified to co-precipitate with SNHG15 that contain DDX5 and RTF1. ( E , F ) DDX5 and RTF1 antibodies were used to immune-precipitate their associated RNA targets in the nuclear extracts of breast cancer cells. Normal IgG was utilized in negative controls. SNHG15 was co-precipitated with DDX5 or RTF1.

Article Snippet: DDX5 and GPDH antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Expressing, Construct, Stable Transfection, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: SNHG15 Mediates MTSS1 Gene Expression via Interacting with the Gene Promoter and Regulating Transcription Pausing

doi: 10.3390/ijms252111565

Figure Lengend Snippet: Roles of DDX5 and RTF1 in SNHG15-mediated MTSS1 mRNA expression. ( A ) qRT-PCR indicated that DDX5 mRNA was silenced by siRNA treatment. ** p < 0.01. ( B ) Levels of SNHG15 were decreased after knocking down of DDX5 mRNA. ** p < 0.01. ( C ) The role of DDX5/SNHG15 in MTSS1 mRNA expression was determined in SNHG15-expressing BT549 cells. Knocking down either SNHG15 or DDX5 increased expression of MTSS1 mRNA. ** p < 0.01. ( D ) DDX5-RIP was performed to detect which region of SNHG15 was binding to DDX5. The results showed that the T1 truncate or 5′ region of SNHG15 was associated with DDX5. ** p < 0.01. ( E ) RTF1-RIP was performed to detect the region of SNHG15 that bound to RTF1. The results showed that the T2 truncate or 3′ region of SNHG15 bound to DDX5. ** p < 0.01. ( F ) Effect of RTF1 on MTSS1 mRNA expression was determined by silencing of RTF1 with siRNA (upper). Knocking down of RTF1 moderately increased MTSS1 levels (lower). * p < 0.05, ** p < 0.01.

Article Snippet: DDX5 and GPDH antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: SNHG15 Mediates MTSS1 Gene Expression via Interacting with the Gene Promoter and Regulating Transcription Pausing

doi: 10.3390/ijms252111565

Figure Lengend Snippet: A model of SNHG15-mediated repression of MTSS1 transcription. Step 1: In the nucleus, SNHG15 complexes with DDX5 and RTF1. The middle part of SNHG15 associates with DDX5 and its 3′ region binds to RTF1. Step 2: Subsequently, the 5′ region of SHNG15 interacts with the core promoter of the MTSS1 gene. This interaction not only changes the architecture of the DNA promoter but also allows SNHG15 to transport its 3′-associated RTF1 to the core promoter and contact with RNA Pol II. Step 3: Binding of SNHG15 to the core promoter interferes with MTSS1 transcription while interaction of RTF1 with RNA Pol II regulates the pausing process, thus resulting in repression of MTSS1 transcription.

Article Snippet: DDX5 and GPDH antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Binding Assay