Journal: Journal of Hepatocellular Carcinoma

Article Title: m6A-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating BMPR1A Transcription

doi: 10.2147/jhc.s480522

Figure Lengend Snippet: Figure 8 m6A-methylated NUTM2B-AS1 bound and recruited YTHDC2 and MLL1 to BMPR1A promoter region to activate BMPR1A expression. (A) RIP assays were performed to detect the binding of NUTM2B-AS1 to YTHDC2 and MLL1 in SNU-398 cells with overexpression of METTL3 or METTL16. (B) RIP assays were performed to detect the binding of NUTM2B-AS1 to YTHDC2 and MLL1 in SNU-398 cells with depletion of METTL3 or METTL16. (C) ChIP assays were performed to detect the binding of YTHDC2 and MLL1 to BMPR1A promoter region in SNU-398 cells with overexpression of WT or mutated NUTM2B-AS1. (D) ChIP assays were performed to detect the binding of YTHDC2 and MLL1 to BMPR1A promoter region in SNU-398 cells with depletion of NUTM2B-AS1. (E) BMPR1A expression was measured by qPCR in SNU-398 cells with concurrent overexpression of NUTM2B-AS1 and depletion of YTHDC2. (F) BMPR1A expression was measured by qPCR in SNU-398 cells with concurrent overexpression of NUTM2B-AS1 and depletion of MLL1. (G and H) The correlation between BMPR1A and YTHDC2 (G) or MLL1 (H) expression level in 371 hCC tissues, according to TCGA LIHC data. r and P values were calculated by Spearman correlation analysis. For (A-F), results are shown as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by one-way ANOVA followed by Dunnett’s multiple comparisons test (A-D) or Student’s t-test (E and F).

Article Snippet: RNA Immunoprecipitation (RIP) and Methylated RNA Immunoprecipitation (MeRIP) Assays The binding of RNA to proteins was detected using the RIP assay, which was conducted in SNU-398 cells using an EZMagna RIP Kit (cat. no. 17–701, Millipore, Billerica, MA, USA) and antibodies against YTHDC2 (cat. no. ab220160, Abcam, Cambridge, MA, USA) or MLL1 (cat. no. 34907; Cell Signaling Technology, Danvers, MA).

Techniques: Methylation, Expressing, Binding Assay, Over Expression

Journal: European journal of human genetics : EJHG

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome.

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Fig. 2 Facial characteristics of the ID patients carrying a KMT2A variant. Patient 2 (P2) carries the c.8558T>G (p. (Met2853Arg)) variant, Patient 3 (P3) carries the c.3581G>A (p. (Cys1194Tyr)) variant and Patient 4 (P4) carries the c.11322–1G>A variant

Article Snippet: We used KMT2A (NM_005933) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL SiteDirected Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Variant Assay

Journal: European journal of human genetics : EJHG

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome.

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Fig. 4 Mapping nuclear targeting signals of wild-type (WT) and mutated KMT2A. Wild type or mutated KMT2A constructs (c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg))) were transiently transfected into COS7 cells and detected by staining with anti-MLL-1 (KMT2A) antibody. a Representative examples of typical patterns; uniform pattern and dot patterns (small dots or bigger patches absent within the nucleoli). b Distribution (% ± SEM) of the different nuclear patterns of cells expressing wild-type or mutated KMT2A constructs. Results were obtained by using data from more than 600 transfected cells of each construct in four independent experiments. The KMT2A c.3460C>T (p.(Arg1154Trp)) mutant abolishes sig- nificantly its capability to produce big dots (***χ2 test with p < 0.0001)

Article Snippet: We used KMT2A (NM_005933) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL SiteDirected Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Construct, Transfection, Staining, Expressing, Mutagenesis

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: 3D-structure predictions for APT1-MLL1 interactions. (A) Predicted 3D-structure of APT1 was constructed. The numbers represent the order of nucleotide residues. Some nucleotide residues are indicated. (B) 3D-structure of the complex was calculated using NPDock web server and illustrated in two different views. MLL1 SET domain is represented as subdomains with different colors such as the N-flanking region in red, SET-N in dark yellow, SET-I in green, SET-C in cyan, and post-SET in blue. (C) Hydrogen bonds formed between APT1 and MLL1 protein in two regions (dotted lines). The bases interacting with MLL1 amino acids are illustrated.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Construct

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Evaluation of enrichment of MLL1-binding aptamers during SELEX. (A) Binding activity was measured using various amounts of S0, S3, and S16 libraries and 1 μg of MLL1 protein. The binding activity was calculated by qRT-PCR as described in “Materials and Methods”. Data are presented as means ± SEM. (B) Sequencing data of S0, S3, and S16 libraries were obtained using NGS. The percentage population of the five most popular abundant aptamers was revealed. (C) Sequence abundance in the sub-population consisting of the top 50 sequences. The percentage in the population of top 50 unique sequences was determined from S1, S3, and S16 libraries.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Binding Assay, Activity Assay, Quantitative RT-PCR, Sequencing

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Evaluation of binding affinity of aptamers. (A) 1 μg of APT1, APT2, APT3, and three libraries were incubated with 1 μg of MLL1 protein and the binding activity was determined by qRT-PCR analysis. The percentage of bound RNA was expressed. (B) Mutation or deletion was introduced in APT1 as illustrated. The randomized region of APT1 are enclosed in a box, mutated nucleotides are underlined, and the deleted nucleotides are marked with hyphen. (C) The binding activity of each variant (1 μg) was measured by qRT-PCR analysis and the amount of bound RNA was calculated. Relative amount of bound RNAs was expressed as a fold change using wild-type APT1 was used as a control.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Binding Assay, Incubation, Activity Assay, Quantitative RT-PCR, Mutagenesis, Variant Assay

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Effect of aptamers on MLL1 activity. MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit. S-adenosylmethionine was added as a methyl-group donor. MLL1 (2 ng/μl) was incubated with or without aptamers. S0 library was used as a control. * p <0.05, *** p <0.005 versus control. Data are presented as means ± SEM. Statistical significance was determined with unpaired Student’s t -test.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, Incubation