Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 and TG003 chemical structures. ( b ) Pharmacokinetic profile of TG693 after a single 30 mg kg −1 dose administered by subcutaneous injection in imprinting control region (ICR) mice. Data indicate the mean ± SEM (n = 3). ( c ) Recombinant CLK1 was incubated with the substrate peptide in the presence of the indicated concentrations of small molecules. Data represent the means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) TG693 competitive ATP inhibition is shown in Michaelis-Menten (left) and Hanes-Woolf (right) plots. CLK1 kinase activity was measured at the indicated concentrations of TG693 and ATP. Velocity was plotted versus [ATP] and [ATP]/velocity was plotted versus [ATP]. ( e ) Map of the inhibitory activities of TG693 on a kinase dendrogram. Percent inhibition by 1 μM TG693 was measured for a panel of 313 kinases. Red circles indicate the inhibited kinases and are sized according to percent inhibition. The illustration was reproduced courtesy of Cell Signaling Technology, Inc. ( www.cellsignal.com ).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Injection, Recombinant, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) SR protein phosphorylation was assessed in HeLa cells treated with TG693 and TG003 for 1 h. Lamin B served as a loading control. Uncropped images have been provided in . ( b , c ) Effect of TG693 on exon 31 skipping with the reporter plasmid. Transfected HeLa cells were incubated in the presence of TG693, TG003, or DMSO vehicle for 24 h. Reporter and endogenous CLK1 splicing was then analyzed by RT-PCR. GAPDH served as a control. The Splicing ratios were quantified by intensity analysis and normalized to GAPDH expression. Uncropped images have been provided in and , respectively. Data represent the means ± SD (n = 3).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Plasmid Preparation, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 bioavailability in the tibialis anterior (TA) muscle of ICR mice after oral administration of a single 30 mg kg −1 dose. Data represent the mean ± SEM (n = 3). ( b ) SR protein phosphorylation status in the TA muscle of ICR mice after oral administration. Lamin B served as a loading control. SRSF4 phosphorylation was quantified by densitometry. Uncropped images are provided in . Data represent means ± SD (n = 5). * p < 0.05. ( c,d ) Clk1 expression in the TA muscle, heart and diaphragm were analyzed by RT-PCR with a GAPDH internal control. Uncropped images are provided in and in , respectively. Data represent the means ± SD (n = 3). * p < 0.05.

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a WB analysis and quantification of CDK9 protein levels in MV4–11 cells treated with 200 nM DbTACs with different linker lengths (DbTACs-8, −11, −16, −21, −26, and −57 Å) and a positive compound B11 for 6 h. GAPDH was used as a loading control. An unpaired two-tailed t -test was used to evaluate statistical significance. **** P < 0.0001 (Control vs. DbTACs-26 Å), n.s. represents no significance. The error bars indicate the mean ± SD values; n = 3. b Immunofluorescence staining images of human hepatoma cells (HepG2) treated with 200 nM DbTACs with different linker lengths or control for 6 h. The nuclei were stained with DAPI in blue, and CDK9 protein was stained in green. The red dotted square in the merged layer indicates an individual cell at a higher magnification. Scale bars, 20 and 3 μm, respectively. c Concentration-dependent degradation and d time degradation characteristics of CDK9 by representative DbTACs-26 Å analyzed by WB. GAPDH was used as a loading control. An unpaired two-tailed t -test was used to evaluate statistical significance. ** P = 0.0083, *** P = 0.0009, **** P < 0.0001 (Control vs. 6 h+ and Control vs. 12h+). The error bars indicate the mean ± SD values; n = 3.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Control, Two Tailed Test, Immunofluorescence, Staining, Concentration Assay

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Live-cell imaging was performed to visualize the real-time localization of CDK9 in HEK293T cells and to track the decrease in CDK9 after treatment with DbTACs-26 Å for 6 h. The scale bars, 40 μm. b SEC-HPLC analysis of retention time of DbTACs-26 Å after incubation with human recombinant CDK9 or CRBN protein or both. c Molecular docking sites of the ternary complex in an all-atom model. SPR sensorgrams were employed to monitor the interaction between e DbTACs-26 Å (binary complexes) or d DbTACs-26 Å, f DbTACs-8 Å, and g DbTACs-57 Å preincubated with human recombinant CRBN protein (ternary complexes) and immobilized CDK9 protein. h WB analysis of the selective degradation potency of DbTACs-26 Å in MV4–11 cells. The cells were co-incubated with different treatments and collected after 6 h. i A ligand competition test for the degradation of the target protein by CDK9 degrader DbTACs-26 Å. Inhibitors of CDK9, CRBN, and proteasome (BAY., P.M., and MG132, respectively) were used, and all signals of each band were normalized successively to GAPDH.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Live Cell Imaging, Incubation, Recombinant

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Volcano plot showing fold changes of protein abundance from global proteomics analysis of MV4–11 cells treated with DbTACs-26 Å for 6 h at 200 nM. Statistical test ( t -test analysis). b Subcellular localization prediction of identified proteins using WoLFPSORT. The subcellular localization of identified proteins was predicated using the WoLFPSORT database with amino acid sequences of identified proteins. c Gene ontology (GO) analysis of a cellular component of samples between DbTACs-26 Å-treated group and control group. Statistical test (Fisher’s exact test). Molecular function analysis of compound DbTACs-26 Å-treated group and control group. Cluster analysis of d biological process, e molecular function, and f KEGG pathway of samples between DbTACs-26 Å-treated group and control group. n = 3. Statistical test (Fisher’s exact test). g Protein–protein interaction network analysis of CDK9 with other proteins.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Quantitative Proteomics, Control

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Schematic illustration of bis-DbTACs design, which is based on DbTACs. b Schematic illustration of three ligand covalent sites of bis-DbTACs equivalent to a DNA tetrahedral scaffold with three polyA domains. Au NPs (5, 10, and 15 nm) correspond to CRBN, CDK9, and CDK6 ligands, respectively. c Cartoon and representative TEM images of bis-DbTACs equivalents. Scale bars are 75 Å and 200 Å, respectively. d WB analysis of the selectively targeted degradation ability of bis-DbTACs at different concentrations and semiquantitative analysis of the grayscale. The error bars indicate the mean ± SD values; n = 3. e Immunofluorescence double-staining images of HepG2 cells treated with/without bis-DbTACs were recorded by confocal laser scanning microscopy. The cell nucleus was stained with DAPI. CDK6 and CDK9 proteins were labeled with anti-CDK6 and anti-CDK9 antibodies, respectively. Scale bars, 10 μm.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Immunofluorescence, Double Staining, Confocal Laser Scanning Microscopy, Staining, Labeling

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Strategy for designing Abs-DbTACs using CDK9 antibody as the POI ligand. b Self-assembly process of Abs-DbTACs was analyzed by agarose gel electrophoresis. The preparation of Abs-DbTACs was verified by c UV‒visible spectra and d SEC-HPLC. e WB analysis of the targeted CDK9 degradation ability of Abs-DbTACs at different concentrations in MOLM13 cells. A free CDK9 antibody was chosen as the negative control (NC). A semi-quantitative analysis of their grayscale values was performed. The error bars indicate the mean ± SD values; n = 3. An unpaired two-tailed t -test was used to evaluate statistical significance. ** P = 0.0016, *** P = 0.0002, **** P < 0.0001 (Control vs. 191 nM Abs-DbTACs and Control vs. 497 nM Abs-DbTACs), n.s. represents no significance.

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1-AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Agarose Gel Electrophoresis, Negative Control, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Host-pathogen interactions in the Plasmodium -infected mouse liver at spatial and single-cell resolution

doi: 10.1038/s41467-024-51418-2

Figure Lengend Snippet: a Schematic representation of the experimental design of this study. Livers were collected at 12, 24, or 38 h post infection (hpi) with P. berghei parasites or salivary gland lysate of uninfected mosquitoes (SGC) (left). Immunofluorescence (IF) staining of the parasite, spatial transcriptomics (ST) or 10X visium spatial technology protocols, and droplet-based single-nuclei RNA sequencing (snRNA-seq) were performed (center). Both data were further analyzed computationally (right). IHS stands for immune hotspot. b After dimensionality reduction, the normalized and batch-corrected data were embedded in UMAP space and split by the original condition for visualization. Data from SGC sections are shown on the top from 12 to 38 hpi (left to right) and data from P. berghei - infected sections are shown on the bottom from 12 to 38 hpi (left to right). Clusters with an obvious association to infection conditions are highlighted with gray boxes. c For identified clusters ST10 and ST11, differential gene expression analysis (DGEA) was performed, followed by functional enrichment analysis for each cluster (see Methods for details). Overrepresented pathways of the KEGG database for ST10 are shown in rose and for ST11 in aquamarine. Scales for expression values of overrepresented genes belonging to the individual KEGG pathways are shown for ST11 (left) or ST10 (right), from high expression (dark) to lower expression (light). Selected gene names are shown at the bottom. Enrichment scores for the pathways are shown on the right. d Interaction analysis of clusters was performed to evaluate spatial enrichment expression programs as suggested by clustering analysis in space. Positive enrichment values (orange) indicate spots belonging to these clusters are more likely to be neighboring, while negative enrichment values (blue) indicate spots associated with these expression programs are less likely to be neighboring. Clusters without significant enrichment in each other’s neighborhoods are shown in white. e Visium clusters were imposed on spatial positions and annotated according to spatial expression features. Sections of the investigated conditions are divided for ease of inspection as in ( b ), with the top panel comprising SGC sections across 12–38 hpi and the bottom panel comprising P. berghei infected sections across 12–38 hpi.

Article Snippet: In this study, we use a combination of the original Spatial Transcriptomics 2K arrays , (henceforth referred to as ST) and Visium arrays (10X Genomics Inc.) .

Techniques: Infection, Immunofluorescence, Staining, RNA Sequencing, Gene Expression, Functional Assay, Expressing

Journal:

Article Title: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse

doi:

Figure Lengend Snippet: Efficient nuclear uptake of liposome-complexed fluorescein-labeled AO by H-2Kb-tsA58 mdx cells. Cultured H-2Kb-tsA58 mdx myotubes were assessed for uptake of fluorescence after exposure to complexes of Lipofectin and AO 5′SS-FITC (2:1 ratio). Nuclear fluorescence was observed in ≈100% of the myotubes 3 h after transfection, with some pinpoint foci of fluorescence located in the cytoplasm and at the cell surface (B). Many of the transfected myotubes displayed multiple fluorescent nuclei (A).

Article Snippet: H-2K b -tsA58 mdx cells were transfected 48 h after trypsin treatment in a final volume of 0.5 ml of antibiotic- and serum-free Opti-MEM (Life Technologies).

Techniques: Labeling, Cell Culture, Fluorescence, Transfection

Journal:

Article Title: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse

doi:

Figure Lengend Snippet: AO-induced exon skipping in H-2Kb-tsA58 mdx myoblasts. Total RNA was extracted from treated and untreated H-2Kb-tsA58 mdx cells and amplified by nested RT-PCR using primers annealing to exons 20 and 26. Transfections were carried out as described in the text with the indicated AOs. The 901-bp full-length transcript was detected in all samples except the PCR negative (-ve) control. Lane M, size markers. A shorter product of 688 bp (arrow), corresponding to the removal of exon 23, was amplified from cell extracts transfected with AO 5′SS-FITC and AO 5′SS-25.

Article Snippet: H-2K b -tsA58 mdx cells were transfected 48 h after trypsin treatment in a final volume of 0.5 ml of antibiotic- and serum-free Opti-MEM (Life Technologies).

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Primers used in quantitative polymerase chain reaction analysis.

Article Snippet: Primary antibodies against the following proteins were used: BRD4 (1:1,000; cat. no. ab128874), B-Raf proto-oncogene serine/threonine kinase (BRAF; 1:1,000; cat. no. ab33899), ras homolog family member A (RHOA; 1:1,000; cat. no. ab54835), mitogen-activated protein kinase 8 (MAPK8, also known as JNK1; 1:2,000; cat. no. ab54835), MAPK10 (also known as JNK3; 1:1,000; cat. no. ab87404) (all from Abcam, Cambridge, UK), KRAS (1:1,000; cat. no. 12063-1-AP; ProteinTech) and GAPDH (1;10,000; cat. no. SAB2100894; Sigma-Aldrich; Merck KGaA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Experimental validation of microarray results. (A) Reverse transcription-quantitative polymerase chain reaction results for the mRNA expression levels of the ten key genes identified by global signal transduction network analysis. (B) Western blotting validation of the protein expression changes of key genes in the BRD4-shRNA and the Scr-shRNA groups. GAPDH was used as an internal control. (C) Representative photographs and quantification of KRAS immunostaining in normal brain tissue and glioma tissues of grades II, III and IV. Scale bar, 20 µ m. (D) Western blot analysis of KRAS levels in HA and U251 cells. (E) KRAS silencing following siRNA transfection in U251 cells was confirmed by western blotting (at 72 h post-transfection). (F) A cell counting kit-8 assay was performed to detect the proliferation rates of siKRAS and or siCon-transfected U251 cells. Each experiment was performed in triplicate. (G) The apoptosis rates of siKRAS and siCon-transfected U251 cells were determined by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Scar bar, 50 µ m. Experimental data are presented as the mean ± standard deviation of at least three experiments. * P<0.05 and ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; KRAS, KRAS proto-oncogene GTPase; HA, human astrocytes; si, small interfering; Con, control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; OD, optical density.

Article Snippet: Primary antibodies against the following proteins were used: BRD4 (1:1,000; cat. no. ab128874), B-Raf proto-oncogene serine/threonine kinase (BRAF; 1:1,000; cat. no. ab33899), ras homolog family member A (RHOA; 1:1,000; cat. no. ab54835), mitogen-activated protein kinase 8 (MAPK8, also known as JNK1; 1:2,000; cat. no. ab54835), MAPK10 (also known as JNK3; 1:1,000; cat. no. ab87404) (all from Abcam, Cambridge, UK), KRAS (1:1,000; cat. no. 12063-1-AP; ProteinTech) and GAPDH (1;10,000; cat. no. SAB2100894; Sigma-Aldrich; Merck KGaA).

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transduction, Western Blot, shRNA, Control, Immunostaining, Transfection, Cell Counting, TUNEL Assay, Staining, Standard Deviation