Unc1999

10μM UNC1999 (Cayman Chemical) was used to inhibit Ezh2 and 10μM cytochalasin B (Sigma) was used to inhibit actin polymerization. 'mCherry2XNLS-P3D-actin-62R1-pmCherry' (addgene), which expresses nuclear-targeted non-polymerizing R62D mutant of human actin, with an mCherry expressing reporter, was used to specifically inhibit the polymerization of nuclear actin. The plasmid Flag-NLS-actinWT was used as a control (Baarlink et al., 2017 (link); Belin et al., 2015 (link)). The plasmids were transcribed in-vitro and the mRNAs were electroporated (300V, 1ms) into 18hr-differentiated Th2 cells 6hr before harvesting.

Cell culturing, luciferase silencing, and transfections of the Luc14 and Gal4EED/luc HEK293 cell lines were performed as described in Daer et al. 2017. Briefly, cells were transfected in 12-well plates with Lipid-DNA complexes [500 ng plasmid, 3 μl Lipofectamine LTX, 1 μl Plus Reagent (Life Technologies)]. EZH2 inhibition was carried out with UNC1999 (Cayman Chemical) dissolved in DMSO to 10 mM. Gal4EED/luc cells were treated for four days with 1 μg/μl doxycycline to induce silencing. Cells were grown in dox-free media for one day and then treated with 10 μM UNC1999 by diluting 1 μl of 10 mM UNC1999 per 1 ml media for three days. Control cells were treated with 1 μl of DMSO (vehicle) per 1 ml media. Cells were grown for one day in media without UNC1999 prior to luciferase assays, transfection with Cas9/sgRNA-expressing plasmids (described above), or ChIP-qPCR.