Journal: The Journal of Cell Biology

Article Title: L1 retrotransposon antisense RNA within ASAR lncRNAs controls chromosome-wide replication timing

doi: 10.1083/jcb.201707082

Figure Lengend Snippet: Junctions and replication timing phenotypes of HTD114-derived cell clones with CRISPR/Cas9-mediated disruptions in ASAR6 L1PA2, chromosome 6: 96,206,263–96,212,288 (Feb 2009 [GRCh37/hg19])

Article Snippet: Using Lipofectamine 2000 according to the manufacturer’s recommendations, we cotransfected HTD114 cells with plasmids encoding GFP, sgRNAs, and Cas9 endonuclease (Origene).

Techniques: Clone Assay, CRISPR

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Promotion of β-catenin/Foxo1 signaling ameliorates renal interstitial fibrosis.

doi: 10.1038/s41374-019-0276-z

Figure Lengend Snippet: Fig. 2 β-catenin/TCF and β- catenin/Foxo1 interactions in C1.1 cells. a Representative blots of co-IP for β-catenin or IgG (as a negative control) with TCF or Foxo1 in C1.1 cells. RhTGF-β1 (3 ng/ml) increased β-catenin/TCF binding (1st row, 2nd lane) while rhTGF-β1 and ICG-001 (5 and 10 µM) promoted β-catenin/Foxo1 binding (3rd row, 3rd and 4th lane). Western blots of β-catenin in C1.1 cells of control, rhTGF- β1, rhTGF-β1 + ICG-001 (5 µM), rhTGF-β1 + ICG-001 (10 µM), ICG-001 only. Representative of three independent experiments. b Association of β-catenin/ Foxo1 (red spot) and β-catenin/ TCF (green spot) were demonstrated by PLA in C1.1 cells. Nuclei were stained with DAPI. Representative of three independent experiments. Original magnification, X60. Scale bar, 10 µm. c, d Quantitation of PLA spots (fold change) in C1.1 cells by one- way ANOVA followed by Tukey post hoc test. The relative PLA spots of control were arbitrarily set as one. e The relative Foxo reporter luciferase activities are shown in control C1.1 cells, and F-TrCP-Ecad- transfected, rhTGF-β1 treated, rhTGF-β1 and ICG-001 treated, F-TrCP-Ecad transfected and rhTGF-β1 + ICG-001 treated, ICG-001 alone treated C1.1 cells. f β-catenin/TCF activity shown by TOP-Flash assay in C1.1 cells with the various treatments described. Data are representative results of at least three independent experiments performed in triplicate. Quantitation of relative luciferase activity is shown as fold increase in luciferase activity. The control was arbitrarily defined as one. Results are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Generation of Foxo1 KO cell line and TCF1 KO cell line by CRISPR/cas9 To generate Foxo1 KO C1.1 cell lines, we co-transfected Foxo1 CRISPR/cas9 KO plasmid with a Foxo1 HDR plasmid (Santa Cruz Biotechnology, Inc), following the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Binding Assay, Western Blot, Control, Staining, Quantitation Assay, Luciferase, Transfection, Activity Assay