Ezh2 inhibitor gsk126

Cells were transfected with miRIDIAN microRNA has-miR-204-5p mimics or inhibitors or corresponding control oligos (Dharmacon, Lafayette, CO, USA), or SNAI2, SUZ12, HDAC1, STAT3 siRNA or control siRNA (ON-TARGETplus SMARTpool siRNA, Dharmacon,) by using Lipofectamine RNAiMax (Invitrogen) as per the manufacturer's instructions. For plasmid transfection, Lipofectamine® 3000 Transfection Reagent (Invitrogen) was used according to manufacturer's instructions. After culturing for 48 h, transfected cells were harvested for the following studies. For drug treatment, cell lines were treated for 24 h with the following inhibitors: EZH2 inhibitor GSK126 (Cat#S7061, Selleck Chemicals, Houston, TX, USA) at 5 μM and HDAC inhibitor Vorinostat (suberoylanilide hydroxamic acid, SAHA, Cat#S1047, Selleck Chemicals) at 3 μM.

For immunofluorescence microscopy, 4T1 sg-Ctrl and Nup210 KO cells were seeded onto 4-well μ-Slides at a seeding density of 25,000 cells per well. After 24 h, cells were treated with dimethyl sulfoxide, 5 μM EZH2 inhibitor GSK126 (Selleckchem), or 5 μM H3K27me3 demethylase inhibitor GSKJ4 (Selleckchem) for another 24 h. Cells were then fixed with −20 °C methanol for 2 min. Histone H3.1/3.2 and H3K27me3 antibodies were used for immunofluorescence staining according to the protocol described above^{28 (link)}.
For the qRT-PCR analysis, 2 × 10⁵ 4T1 Nup210 KO cells were seeded onto 6 cm dishes. After 24 h, 5 μM GSK126 or 5 μM GSKJ4 was applied to the cells that were then incubated for another 24–48 h. Cells were then lysed, RNA was isolated, and qRT-PCR was performed as described above.