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 above28 (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.
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.
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