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. 2009 Sep 8;284(45):30933–30940. doi: 10.1074/jbc.M109.051441

FIGURE 3.

FIGURE 3.

p16INK4A-induced G0/G1 arrest is associated with repression of BCL2, MCL1, and Noxa and induction of Puma. A, CEM/Ctr, CEM-1D2/p16, and CEM-6E2/p16 cells were treated with 250 ng/ml Dox for 24 and 48 h. The lysates were subjected to immunoblot analyses using specific antibodies directed against BCL2, Bcl-xL, Bcl-w, MCL1, BAX, Bak, Bim, Puma, Noxa, and α-tubulin. B, for RT-PCR analysis, RNA was prepared from CEM/Ctr, CEM-6E2/p16, and CEM-1D2/p16 cells after treatment with 250 ng/ml Dox for 24 h. mRNA steady state levels of Puma and BCL2 were determined by quantitative real time RT-PCR. The bars represent fold-induction over control (untreated cells) of three independent experiments each performed in triplicate. C, mitochondrial (mito) and cytoplasmic (cyto) extracts were prepared from CEM/Ctr, CEM-6E2/p16, and CEM-1D2/p16 cells cultured in the absence or presence of 250 ng/ml Dox for 24 h. The expression of Puma, BCL2, Noxa, MCL1, and α-tubulin in subcellular fractions was detected by immunoblot.