Journal: Advanced Science

Article Title: GLTSCR1 Negatively Regulates BRD4‐Dependent Transcription Elongation and Inhibits CRC Metastasis

doi: 10.1002/advs.201901114

Figure Lengend Snippet: GLTSCR1 regulates transcriptional elongation and enhances CRC cell sensitivity to BET inhibitors A) Luciferase reporter assay to detect the transcriptional activity of SLC2A1 and SLC2A3 in GLTSCR1‐KO HCT116 cells. B) ChIP‐PCR to detect the DNA binding capacity of Flag‐GLTSCR1 to SLC2A1 and SLC2A3 gene in scramble + empty vector, scramble + Flag‐GLTSCR1, and shBRD4 + Flag‐GLTSCR1 cells through pull‐down by anti‐Flag. C) ChIP‐PCR to detect the DNA binding capacity of BRD4 to SLC2A1 and SLC2A3 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐BRD4. D) Detection for nascent RNA of SLC2A1 and SLC2A3 in mock and GLTSCR1‐KO cells through RNA pull‐down by anti‐BrUTP. E) Transcription efficiency of SLC2A1 and SLC2A3 in GLTSCR1‐KO HCT116 cells, as determined by RT‐qPCR. F) ChIP‐PCR to detect the RNA Pol II chromatin occupancy in multiple sites of SLC2A1 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐RNA Pol II (left) and to analyze the accumulation of RNA Pol II in gene body of SLC2A1 after releasing from DRB‐inhibition at 0, 20, and 40 min in mock and GLTSCR1‐KO HCT116 cells (right). The upper schematic diagram represents the primers sites of SLC2A1. G) ChIP‐PCR to detect the RNA Pol II chromatin occupancy in multiply sites of SLC2A3 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐RNA Pol II (left) and to analyze the accumulation of RNA Pol II in gene body of SLC2A3 after releasing from DRB‐inhibition at 0, 20, and 40 min in mock and GLTSCR1‐KO HCT116 cells (right). The upper schematic diagram represents the primers sites of SLC2A3. H) Relative mRNA expression (up) and Immunoblotting analysis (down) of SLC2A1 and SLC2A3 in HCT116 cells transfected with siSLC2A1 and siSLC2A3. I) Transwell assay to investigate the migratory and invasive properties of HCT116 cells transfected with siSLC2A1 and siSLC2A3. The histogram on the right shows the quantification analysis results. J) JQ1 and I‐BET inhibition efficiency in control (mock), GLTSCR1‐KO and GLTSCR1‐C7/C9 heterozygous mutated HCT116 cells. Data are presented as the mean ± SD; statistical significance was assessed by an unpaired t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001; n = 3.

Article Snippet: 50 µL Magnetic beads (Dynabeads Protein G, 10004D, Invitrogen) and 10 µg (20 µL) of anti‐BrUTP monoclonal antibodies (BD Biosciences, 555627) and aliquot RNA were used to pull down the BrUTP‐labeled nascent RNA, which was ready for further RT‐qPCR.

Techniques: Luciferase, Reporter Assay, Activity Assay, Binding Assay, Plasmid Preparation, Quantitative RT-PCR, Inhibition, Expressing, Western Blot, Transfection, Transwell Assay

Journal:

Article Title: A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II

doi:

Figure Lengend Snippet: SCAF8 is associated with transcription sites. Mouse Swiss 3T3 cells were permeabilized and incubated with BrUTP to label transcription sites in situ (Materials and Methods). Transcription sites were detected with rat anti-BrUTP antibody (Sera-Lab) and visualized with green (A). SCAF8 was detected with MAb F1E1 and then visualized with red (B), and the merged images are displayed in panel C. The boxed area, including both extranucleolar and nucleolar regions, is further enlarged and displayed in panels D (transcription sites), E (SCAF8), and F (merged images). The line through F indicates the boundary between the nucleolus (left) and the nucleus (right).

Article Snippet: Transcription sites were detected with rat anti-BrUTP antibody (Sera-Lab) and visualized with green (A).

Techniques: Incubation, In Situ