FIGURE 1

The treatment of a stable AID-EWSR1/AID-EWSR1 DLD-1 cell line with Auxin induces an efficient degradation of AID-EWS. (A). Schematic diagram of OsTIR1 and AID-EWSR1 DNA construct. OsTIR1: plant driven E3 ligase, Bla ^r : Blasticidine resistance gene, Hyg ^r : Hygromycin resistance gene, mAID: mini Auxin-Inducible Degron tag, 3XFLAG; three tandem repeat of FLAG tag. The OsTIR1 construct was inserted at the RCC1 locus, and mAID construct was inserted to both EWSR1 loci (homozygous). (B). Representative images of western blotting using anti-FLAG (top panel), anti-EWSR1 (middle panel) and anti-β actin (bottom panel) obtained from AUX- and AUX + cells in the presence of Auxin at given time point (15 h, 18 h and 25 h). (C). Relative intensity of the bands of western blotting visualized with anti-EWSR1 (normalized to the bands with anti-β-actin) obtained from AUX- and AUX + cells (n = 3 experiments). Graph shows the mean of each group with Standard Deviation (SD). (n = 3 experiments). ^**** p < 0.0001 (Two tailed paired t-test). (D). Representative images of immunocytochemistry using anti-FLAG (green) and DAPI (blue) obtained from AUX- and AUX + cells. Scale bar = 10 um.