Journal: Molecular cell

Article Title: KAP1 is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs

doi: 10.1016/j.molcel.2020.04.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-NELFE , Santa Cruz Biotechnology , Cat# sc-377052; lot #L0117.

Techniques: Virus, Mutagenesis, Recombinant, Membrane, Protease Inhibitor, SYBR Green Assay, Western Blot, Staining, Plasmid Preparation, RNA HS Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, Cloning, shRNA, Software

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: Role of the RNA binding protein NELFE in hepatocellular carcinoma cells. (A) Kaplan-Meier survival analysis of LCI and TCGA-LIHC datasets based on segmentation values of NELFE (high: log2 >0.2, low: log2<0.2). (B–H) Cell proliferation rates measured by xCELLigence (B), colony formation (C), oncosphere formation measured by Algimatrix 3D assay (D), cell migration (E), cell invasion (F), cropped immunoblot (G), and proportions of cells in during G2/M phase measured by 7′AAD staining using flow cytometry (H) of Hep3B and Huh1 HCC cells after shNELFE or shCtrl via lentivirus. Statistical significance for the proliferation rate (B) is measured at time-point 72 hr, results shown in (E–H) were measured at 48 hr. For invasion assay (with matrigel), relative invasion index is calculated by normalizing to cells that have migrated (no matrigel). (I) Bioluminescence of NOD/SCID mice at eight weeks (middle panel). On the right panel, mean signal from shCtrl (n=4) or shNELFE (n=6). (J) Representative livers of three mice in each group. Arrows are pointing at tumor nodules. Scale bars, 1 cm. *p<0.05, **p<0.01, ***p<0.001. All data are mean ± SD. See also Figure S2.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: RNA Binding Assay, Migration, Western Blot, Staining, Flow Cytometry, Invasion Assay

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: NELFE enhances MYC tumorigenicity. (A) Bar graph of colony formation assay of HHT4 cells or HHT4 cells ectopically expressing the indicated proteins at day 10. (B) Representatie image and quantifiation of oncosphere formation assay at day 7 measured by Algimatrix 3D assay. Scale bar, 200 μM. (C) Proliferation rates of different cell lines up to 72 hr. (D) RT-PCR analysis of relative mRNA expression of MYC-related genes. (E) Hematoxylin and eosin and immunohistochemical staining of indicated tumors. Scale bars, 40 μM. (F) Number of tumor nodules four weeks aftr the injection of indicated cells. Short horizontal lines represent the mean. (G) RT-PCR analysis of relative mRNA expression of MYC-related genes in MYC or MYC+NELFE tumor tissues. Data were first normalized to β–actin to get dCt. Relative mRNA was then calculated using 2dCt. *p<0.05, **p<0.01. All data are mean ± SD. See also Figure S4.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: Colony Assay, Expressing, Tube Formation Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Injection

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: NELFE affects MYC-related genes by modulating MYC binding. (A) MYC transam assay on HCC cells treated with 48 hr of NELFE siRNA compared to scrm (*p<0.01). (B) ChIP-PCR of MYC-related genes of HCC cells after shNELFE compared shCtrl. Data is relative to 2% input. (C) ChIP-PCR of MYC-related genes of HHT4-SV40 cells overexpressed with Ctrl, NELFE, MYC or MYC+NELFE. Anti-MYC was used for IP. (D) ChIP-PCR of MYC-related genes in Hep3B cells after 48 hr of MYC siRNA or scrm. Anti-NELFE was used for IP. (E) Represented immunoblot of co-IP assay in HCC cells. MYC or Rabbit IgG was used for IP. All data are mean ± SD. See also Figure S6.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay