Figure 3

K222 methylation promotes proteasome-independent degradation of OCT4. (A) NCCIT cells were infected with LSD1 shRNA lentiviruses. After 96 h, cells were pre-treated with 5 μM MG-132 or vehicle for 1 h, followed by addition of 20 μg/ml CHX and further incubation for 0 to 12 h in the presence of MG-132. Whole cell lysates were immunoblotted with the indicated antibodies. The curves represented quantitation of the OCT4 protein levels. (B) NCCIT cells were pre-treated with 100 μM 2-PCPA and 5 μM MG-132 for 1 h and followed by the same steps as described in (A). (C) NCCIT cells pre-treated with DMSO (left panels) or 2-PCPA (right panels) were exposed to CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in (D). (E) U87 cells were transfected with the shOCT4-FLAG-OCT4 plasmids (WT and variants). After 72 h, they were treated with 20 μg/ml CHX for 0 to 24 h, the whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in (F). (G) NCCIT cells were pre-treated with 5 μM MG-132 or 200 μM chloroquine singly or in combination for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0–24 h. Whole cell lysates were immunoblotted with the indicated antibodies. (H) NCCIT cells were pre-treated with 100 μM 2-PCPA alone or in combination with 200 μM chloroquine for 1 h followed by addition of 20 μg/ml CHX and further incubation for 0 to 24 h. Whole cell lysates were immunoblotted with the indicated antibodies, and the OCT4 protein levels were quantified in (I). Results shown in (D) and (F) were presented as means ± S.D. of triplicate measurements from single experiment representative of 3 independent experiments. Two-tailed unpaired Student’s t tests were used for statistical analyses. *P < 0.05 and **P < 0.01. Cropped immunoblot images were presented for (A), (B), (C), (E), (G) and (H).