Loss of PTP expression in apoptotic cells leads to an increase in cell proliferation.

<p>PTP gene expression was silenced in ER-dnE2F1 cells with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total protein was isolated from lysated cells, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Morphological changes in uninduced and induced ER-dnE2F1 cells were compared with morphological changes in induced cells after knockdown of PTPs. (C) The number of cells within three random fields (100X) were counted and results are expressed as the mean + standard deviation. (D) Cell viability was determined by WST-8 assay in two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (*) <i>p</i><0.05; (**) <i>p</i><0.01; (***) <i>p</i><0.001.</p

<i>PTEN</i> transcription is directly regulated by Rb/E2F complexes.

<p>ER-dnE2F1 cells were treated IPTG and with 4-OHT to induce Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total RNA was isolated at the indicated time points following induction of apoptosis and used for qPCR analysis for two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (B) Chromatin was isolated from the cells, sheared and immunoprecipitated with control mouse IgG or E2F1 antibody. A ∼300 bp sequence within the PTEN promoter region was amplified by PCR. GAPDH was used as control. (−) Untreated; (+) Treated; (NA) No antibody negative control; (*) No chromatin negative control.</p

Knockdown of PTP expression leads to a reduction in caspase activity.

<p>ER-dnE2F1 cells were transfected with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 24 or 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Caspase-3/CPP32 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (B) FLICE/Caspase-8 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (C) Total protein was isolated from lysated cells at 24 h following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for cleaved caspase-3 (Denoted by arrow), caspase-3 and β-actin. (*) <i>p</i><0.05; (**) <i>p</i> = 0.01; (***) <i>p</i><0.01.</p

SHP-2 and PTEN protein expression increases during Rb/E2F-associated apoptosis.

<p>ER-dnE2F1 cells were treated with 4-OHT for the indicated times to induce Rb/E2F-associated apoptosis. Control cells (0 h) were left untreated. (A) Total protein was isolated from lysated cells at the indicated time points following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Relative expression levels of PTP-1B, SHP-2, and PTEN were quantified by densitometry. (h) Hours.</p