Figure 2.

Dose-response change of Cas9 protein level by Shield1 ligand

(A–C) H9-Cas9 cells were seeded in six-well plates 24 h before doxycycline and Shield1 ligand addition. (A) The cells were collected 24 h after 300 ng/mL doxycycline and different concentrations of Shield1 ligand addition. After protein and mRNA extraction, Cas9 protein level was measured by Wes assay, and mRNA level was determined by RT-qPCR. Cas9 protein level was regulated by Shield1 ligand in a dose-dependent manner, whereas Cas9 mRNA level was unchanged. (B) The cells were collected at different time points after 300 ng/mL doxycycline and 250 nM Shield1 ligand treatment. Twenty-four hours after doxycycline and Shield1 addition, the medium was replaced with fresh medium without doxycycline and Shield1. Removal of Shield1 results in decrease of Cas9 protein level. (C) Kinetic study for Cas9 protein level with or without doxycycline and/or Shield1 ligand. First row is the Cas9 protein level. Second row is the densitometry data for the first row Cas9 protein level. Third row is the Nanoluciferase activity, which represents the Cas9 mRNA level. Data are shown as mean values ±SEM of triplicate experiments.