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:

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: Nature

Article Title: Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I

doi: 10.1038/s41586-020-2229-5

Figure Lengend Snippet: a,b, Autophagy flux ( a ) and cell surface MHC-I levels in PDAC cells measured by flow cytometry. Mouse PDAC cells expressing the GFP-LC3-RFP reporter and doxycycline (Dox)-inducible mTurquoise2-Atg4B C74A were grown as organoids for 8 days and treated with Dox (1 μg/ml) for the indicated hours. a, Autophagy flux represented by GFP/RFP ratio. Note that increased GFP/RFP ratio indicates reduced autophagy flux. b, Cell surface MHC-I (H-2K ) levels. Representative flow cytometry plots were shown. Data are mean ± s.d. n = 3 biological replicates. Data are representative of at least four independent experiments. c, Fold changes of respective molecules on the cell surface quantified by flow cytometry. HY15549 cells expressing Dox-inducible mTurquoise2-tagged Atg4B C74A were grown as organoids for 8 days and treated ± Dox (1 μg/ml) for 72 hrs. Positive surface expression of each molecule was confirmed using respective isotype controls. Molecules found in immunological synapses are underlined. TFRC, transferrin receptor. Data are mean ± s.d. n = 4 biological replicates. Representative data from two independent experiments are shown. d-f, (Related to , ) Mouse PDAC cells expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4B C74A were grown as organoids and treated ± Dox (1 μg/mL) for 96 hrs. d, Autophagy inhibition was confirmed by immunoblot. mTurquoise2-Atg4B C74A or endogenous Atg4B were detected by anti-ATG4B antibody. e,f, Flow cytometry plots for H-2K ( e ) and H-2K -SIINFEKL ( f ). Representative plots from , are shown. Grey, isotype control. g , Representative flow cytometry plots of the OT-I cells co-cultured with mouse PDAC cells from . h, (Related to ) Quantitative reverse transcription PCR (qRT-PCR) analysis of OT-I cells that were co-cultured with PDAC cells for 48 hrs. Data are mean ± s.d. n = 3 biological replicates. For g, h, Dox(+) or Dox(−) indicates that PDAC cells were grown ± Dox (1 μg/mL) before co-culture. Dox was not added in co-culture. A representative of at least three independent experiments is shown in d-g . P values determined by unpaired two-tailed t -tests ( a,b,h ). **** P < 0.0001. For gel source data of d , see .

Article Snippet: Organoids were dissociated into single cells, which were then incubated with either anti-H-2K b -SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C.

Techniques: Flow Cytometry, Expressing, Inhibition, Western Blot, Control, Cell Culture, Reverse Transcription, Quantitative RT-PCR, Co-Culture Assay, Two Tailed Test

Journal: Nature

Article Title: Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I

doi: 10.1038/s41586-020-2229-5

Figure Lengend Snippet: a,b, Surface H-2K b ( a ) and H-2K b -SIINFEKL ( b ) measured by flow cytometry. Mouse PDAC cells expressing OVA and Dox-inducible mTurquoise2-Atg4B C74A were grown as organoids and treated ± Dox (1 μg/mL) for 96 hrs ( n = 4 per group). c,d, Co-culture of OT-I cells with HY19636 cells shown in a,b. After 48 hrs, OT-I proliferation was measured by CFSE dilution ( c ) ( n = 4 per group) and PDAC cell viability was measured by Cell-Titer Glo ( d ) ( n = 6 per group). e-r, HY15549 cells carrying Dox-inducible mStrawberry (mSt) or mSt-Atg4B C74A (4B) were orthotopically ( e-h, m-r ) or intrasplenically ( i-l ) injected into syngeneic mice (C57BL/6). MHC-I and PD-L1 expression on PDAC cells ( f,g,j,k,p ) and tumour-infiltrating CD8 + T cells ( h,l,n,q ) quantified by flow cytometry. e-h, Orthotopic tumours harvested on day 20 (mSt, n = 8; 4B, n = 7). i-l, Livers harvested on day 15 ( n = 4 per group). Weight of tumours ( e ) and livers ( i ). Flow cytometry analysis ( f-h , j-l ). m, Weight of tumours following treatment with isotype control IgG or neutralizing monoclonal antibody against CD8 ( n = 7 per group). n,o, Tumours in wild type mice (WT) or Batf3 −/− mice (KO) ( n = 8, 4, 8, and 5; left to right). Quantification of CD8 + T cells ( n ). Tumour weight ( o ). p-r, Tumours expressing control shRNA (Scr) or shRNA against B2m ( n = 8 per group) harvested on day 20. Flow cytometry analysis ( p,q ). Tumour weight ( r ). Data are mean ± s.d. ( a-d ) or s.e.m. ( e-r ). n indicates biological replicates ( a-d ) or individual mice ( e-r ). For a-m, p-r , experiments were performed at least twice and representative data of one experiment are shown. P values determined by unpaired two-tailed t -tests.

Article Snippet: Organoids were dissociated into single cells, which were then incubated with either anti-H-2K b -SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C.

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Injection, Control, shRNA, Two Tailed Test

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

Journal: The Journal of experimental medicine

Article Title: Itraconazole targets cell cycle heterogeneity in colorectal cancer.

doi: 10.1084/jem.20171385

Figure Lengend Snippet: Figure 2. Dormant CRC cells are a differentiated cell population. (A) GO pathway analysis of genes down-regulated in LRCs. (B) GO pathway analysis of genes up-regulated in LRCs. (C) GSEA charts of significantly enriched gene sets in LRCs and non-LRCs. (D) GSEA showing enrichment of the Krt20 differentiated gene set in LRCs and the Lgr5 CSC gene set in non-LRCs. (E) RT-PCR histogram showing enrichment of Wnt targets in PTK7High and differentiation markers in PTK7Low. n = 6; mean ± SEM. ***, P < 0.001; *, P < 0.05 by two-way ANOVA. (F) Bright field images of PTK7High and PTK7Low SW948 spheroid cells 5 d after seeding in nonadherent culture. Bars, 100 µm. (G) Histogram of the tumor-initiating cell frequency (TIC) from FACS sorted SW948 and HT55 spheroids. Mean ± SEM. (H) Column scatter plot of xenograft sizes derived from PTK7High and PTK7Low SW948 cells. Mean ± SEM; **, P < 0.01 by unpaired t test. (I) FACS histogram of PTK7 levels in LRCs and non-LRCs derived from CFSE-labeled SW948 and HT55 spheroids. (J) Pie charts of the relative proportions of LRCs and non-LRCs within PTK7High and PTK7Low populations from SW948 spheroids. Size of each chart is proportional to relative numbers of cells present.

Article Snippet: Antibodies for IHC and FACS: AGR2 (Atlas, HPA007912), GDF15 (Atlas, HPA011191), β-catenin (BD Biosciences, 610154), and PTK7 (clone 188B; Miltenyi).

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Labeling

Journal: The Journal of experimental medicine

Article Title: Itraconazole targets cell cycle heterogeneity in colorectal cancer.

doi: 10.1084/jem.20171385

Figure Lengend Snippet: Figure 3. CCS1-LRCs are differentiated yet can revert to a CSC-like state. (A) Fluorescence micrograph of CFSE-labeled PDO (COLO05) 8 d after labeling with CFSE. LRC marked with arrow. (B) Representative FACS density plot of PTK7 levels in CFSE labeled PDOs. (C) Histogram of B showing the majority of LRCs (CFSE+) are differentiated (PTK7Low). (D) FACS histogram showing the sorting strategy for reseeding of SW948 CFSE-labeled spheroid-derived LRCs and non-LRCs. Accompanying fluorescent microscopy images from seeded wells. Bars, 100 µm (A and D). (E) Replating efficiencies of LRCs and non-LRCs across CCSs. (F) FACS quantification of changes in differentiation status (PTK7 levels) in SW948 spheroid LRCs and cycling cells upon replating in adherent culture.

Article Snippet: Antibodies for IHC and FACS: AGR2 (Atlas, HPA007912), GDF15 (Atlas, HPA011191), β-catenin (BD Biosciences, 610154), and PTK7 (clone 188B; Miltenyi).

Techniques: Fluorescence, Labeling, Derivative Assay, Microscopy

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: Fig. 4 | Real-time visualization of protein degradation and mechanism of DbTACs. 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

Article Snippet: The primary antibody used was mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:100), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:100).

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: Fig. 7 | Design, preparation, characterization, and efficacy of Abs-DbTACs formed using antibody as POI ligand. 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

Article Snippet: The primary antibody used was mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:100), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:100).

Techniques: Agarose Gel Electrophoresis