Journal: bioRxiv
Article Title: Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC
doi: 10.1101/2024.06.17.599429
Figure Lengend Snippet: A-C) Schematic representation of the approach used for the generation of homozygous AID-NCL/OsTIR1 cell lines. Multi-allelic editing of endogenous NCL gene was achieved using two independent donor plasmids and a gRNA designed against the first translated codon of the gene ( A ). Selected multi-allelic NCL edited cells ( B ) were further modified to integrate the OsTIR1 transgene into the AAVS1 -safe harbor site ( C ). See also . D ). Western blot of endogenous NCL in engineered clones, in comparison with parental (Par) cells. Anti-GFP antibody was used to detect the OsTIR1 fused to mEmerald. GAPDH was used as loading control. E) NCL degradation by western blot at 24 h upon 5-Ph-IAA treatment in two different MDA-MB-231 clones (C10.C4 and C10.D9) compared with MDA-MB-231 cells only transduced with OsTIR1. Calnexin was used as loading control. F) Diagram of the hierarchical generation of the clones used in the current study. G) Representative images of Incucyte experiments on MDA-MB-231 mCherry2/Halo-AID-NCL/OsTIR1-mEmerald cells. The RFP channel was used to detect mCherry2-AID-NCL, and the GFP channel was used to assess OsTIR1-mEmerald expression and localization. H) Quantitative analysis of mCherry (AID-NCL) intensity from Incucyte experiments on the indicated cell clones. RFP integrated intensity per image was normalized for t=42’ to account for potential plate condensation at the early time points. The experiment was performed in 5 biological replicates, with 5 technical replicates. Error bars show standard deviation. I-J ) Dose-response curve ( I ) and relative magnification ( J ) for mCherry-AID-NCL degradation using the indicated amounts of 5-Ph-IAA. RFP integrated intensity per image was normalized for t=0. The experiment was performed in three biological replicates, with four technical replicates. Error bars show standard deviation.
Article Snippet: Primary antibodies used were anti-NCL (D4C7O) Rabbit mAb (Cell Signaling Technology, #14574); anti-GFP (B-2) Mouse mAb (Santa Cruz Biotechnology, sc-9996); anti-OsTIR1 Rabbit polyclonal Antibody (MBL Bio, #PD048); anti-GAPDH (14C10) Rabbit mAb (HRP Conjugate (Cell Signaling Technology, #12231).
Techniques: Modification, Western Blot, Clone Assay, Comparison, Control, Transduction, Expressing, Standard Deviation
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