FIG 3.

HP1α loss upon EZH2 depletion does not result from reduced HP1α transcription but is due to proteasomal degradation. (A) HT1080 cells were transfected with either siLuci or siEZH2-A and collected 72 h after transfection. Cell extracts were subjected to subfractionation, and total, nucleoplasm, chromatin, and cytoplasm fractions were analyzed by Western blotting using the indicated antibodies. (B) Transcript levels of HP1α gene were measured by qRT-PCR and normalized to β-actin (ACTB) transcripts in HT1080 cells treated for 72 h with two different siRNAs against EZH2 or with siLuci (n = 3 independent experiments). (C) MG132 proteasome inhibitor suppresses HP1α degradation in siEZH2-treated cells. Thirty hours after siRNA transfection, HT1080 cells were incubated with either 3 μM MG132 (+) or dimethyl sulfoxide (DMSO) (−) for 18 h. Total protein levels for HP1α, EZH2, and β-actin were analyzed. (D) Quantification of HP1α protein levels from panel C (n = 3 independent experiments). (E) HP1α levels are also decreased in SUZ12-depleted cells. HT1080 cells were treated with siSUZ12 or siLuci for 6 days and processed as described for panel A.