human renal epithelial cells hek293t  (ATCC)

Journal: Molecular Metabolism

Article Title: The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

doi: 10.1016/j.molmet.2020.100996

Figure Lengend Snippet: Purb acts as a transcriptional repressor to inhibit ApoA4 expression. (A) Western blot detected the ApoA4 and Purb expression levels after knockdown or overexpression of Purb in BRL-3A cells. (B) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated ApoA4 promoters with pcDNA3.1 vector or pc3.1- Purb in HEK293T cells. (C) The schematic diagram shows that Purb binding site (PNR) contained at 2000 bp of ApoA4 promoter was well conserved. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of ApoA4 promoters of 2000 bp with pcDNA3.1 vector or pc3.1- Purb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfected with wt/mutApoA4 promoters of 2000 bp with pc3.1- Purb in HEK293T cells. (F) ChIP analysis of Purb interaction with the ApoA4 promoter. BRL-3A cells lysates were immunoprecipitated with anti- Purb or control mouse IgG antibody. (G–H) Overexpression of lnc19959.2 and control in BRL-3A cells, respectively, were incubated with the proteasome inhibitor MG-132 (10 μM) or the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) for 6 or 12 h. The protein level of Purb extracted from the whole cell was detected by western blot. (I–J) BRL-3A cells lysates were immunoprecipitated with an HA or Flag-specific antibody in BRL-3A cell lysates which were stably expressing ubiquitin with C-terminal HA tag or Purb with C-terminal Flag tag, respectively. And then they were analyzed by western blot with anti- Purb or anti-Ubiquitin. Bottom, the input of the cell lysates. (K) Western blot detects the ubiquitination levels in BRL-3A cells. Unpaired t -test was used to measure the statistical significance; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The BRL-3A rat liver cell line and HEK293T human renal epithelial cell lines (American Type Culture Collection) were cultured in glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) at 37 °C with humidified air and 5% CO 2 .

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, Control, Incubation, Stable Transfection, Ubiquitin Proteomics, FLAG-tag

Journal: Molecular Metabolism

Article Title: The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

doi: 10.1016/j.molmet.2020.100996

Figure Lengend Snippet: Cebpb activates the lnc19959.2 expression. (A) Heatmaps present gene expressions selected from sequence prediction software. (B–C) RT-qPCR analysis of lnc19959.2 expression levels in BRL-3A cells in the condition of knockdown or overexpression of Cebpb. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated lnc19959.2 promoters with pcDNA3.1 vector or pc3.1-Cebpb in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of lnc19959.2 promoters of 1500 bp with pcDNA3.1vector or pc3.1-Cebpb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (F) Schematic diagram shows that the part of lnc19959.2 promoter sequence of wt and mut of Cebpb binding site is located in −1500～-1000 bp. (G) Dual-luciferase reporter assay detected relative luciferase activities after being cotransfected with wt/mutlnc19959.2 promoters of 1500 bp with pc3.1-Cebpb in HEK293T cells. (H) ChIP analysis of Cebpb interacted with the lnc19959.2 promoter. BRL-3A cells lysates were immunoprecipitated with anti-Cebpb or control mouse IgG antibody. Unpaired t -test was used to measure the statistical significance; ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: The BRL-3A rat liver cell line and HEK293T human renal epithelial cell lines (American Type Culture Collection) were cultured in glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) at 37 °C with humidified air and 5% CO 2 .

Techniques: Expressing, Sequencing, Software, Quantitative RT-PCR, Knockdown, Over Expression, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, Control