Journal: Clinical Epigenetics

Article Title: DNA hypermethylation contributes to colorectal cancer metastasis by regulating the binding of CEBPB and TFCP2 to the CPEB1 promoter

doi: 10.1186/s13148-021-01071-z

Figure Lengend Snippet: TFCP2 binds to the core TF-binding region of CPEB1 when it is hypermethylated. a Silver staining of the nucleoproteins identified in DNA pull-down assay under various conditions. p-WT, the non-methylated CPEB1 promoter; p-Me, the hypermethylated CPEB1 promoter; control, magnetic beads without probes; Input, total nucleoproteins extracted from HCT116 cells; M, protein molecular mass marker. b WB detecting immunoreactive CEBPB in the nucleoprotein fraction after DNA pull-down with the anti-CEBPB antibody. The molecular mass of CEBPB is approximately 35 kDa. c EMSA revealed that CEBPB protein was unable to bind to its target sequence in the hypermethylated TF-binding region of CPEB1 ; 50 × cold probe WT, 50-fold concentration of the unlabelled wild-type CPEB1 promoter which was served as the competitor probe; Bio-Probe WT, a biotin-labelled wild-type probe of CPEB1 upstream region; Bio-Probe Mut, a biotin-labelled mutant probe of CPEB1 upstream region; Nucleoprotein, nucleoprotein extracted from HCT116 cells; Me-Bio Probe WT, a biotin-labelled hypermethylated probe of CPEB1 upstream region. d TFCP2 may be a candidate methylation reader at the upstream region of CPEB1 ; Methylation, the hypermethylated CPEB1 upstream region probe; Wild-type, the wild-type CPEB1 upstream region probe; Control, a probe with a scrambled sequence of CPEB1 upstream region; TF, the TFs capable of binding to the CPEB1 upstream as determined by ChIP-Seq. e Competitive EMSA to confirm TFCP2 as a methylation reader for CPEB1 . Bio-probe, a biotin-labelled wild-type CPEB1 upstream region probe; Me-Bio Probe, a biotin-labelled hypermethylated CPEB1 upstream region probe; 50 × Cold Probe, 50-fold concentration of the unlabelled wild-type CPEB1 upstream region probe that served as a competitor of the Bio-probe; 50 × Cold Me-Probe, 50-fold concentration the unlabelled hypermethylated CPEB1 upstream region probe that served as a competitor of the Me-Bio Probe

Article Snippet: Publicly available high-throughput DNA methylation microarray data (Illumina Human Methylation 450 K) of 387 CRC tumours and 45 samples of para-tumour tissue were obtained from the TCGA database (level-3).

Techniques: Binding Assay, Silver Staining, Pull Down Assay, Methylation, Control, Magnetic Beads, Marker, Sequencing, Concentration Assay, Mutagenesis, ChIP-sequencing

Journal: Cancer Science

Article Title: Adoptive cell therapy using tumor‐infiltrating lymphocytes for melanoma refractory to immune‐checkpoint inhibitors

doi: 10.1111/cas.15009

Figure Lengend Snippet: Total mutation burden, transcriptome signature (ssGSEA score), and quantitative real‐time polymerase chain reaction analysis of pretreatment melanoma cells and expanded TILs. A, To evaluate their mutation status, exome sequencing was performed on the melanoma cells used for TIL manufacturing. The numbers of mutations and the mutated genes in each tumor are shown in the upper columns. Transcriptome signatures based on single‐sample gene set enrichment analysis (ssGSEA) of the tumor cells are shown in the lower columns. B, ssGSEA scores of gene sets related to tumor phenotype (based on microarray data) in the tumors of the three melanoma patients. The cut‐off score was set at 4,000. C, D, Quantitative RT‐PCR analysis of the expression levels of chemokines in the primary tumors (C) and of cytotoxic factors in the expanded TIL products (D). The results are fold‐changes in gene expression normalized to the endogenous reference gene; error bars are standard deviations

Article Snippet: Total RNA from isolated melanoma cells was subjected to microarray analysis using an Agilent SurePrint G3 Human GE v3 8x60K Microarray (DNA Chip Research Inc.).

Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Sequencing, Microarray, Quantitative RT-PCR, Expressing