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. Author manuscript; available in PMC: 2010 Jun 4.
Published in final edited form as: Cancer Cell. 2009 Mar 3;15(3):184–194. doi: 10.1016/j.ccr.2009.01.026

Figure 5. pRB facilitates E1A’s ability to promote apoptosis.

Figure 5

(A) In the absence of E1A, a pRB-E2F1 complex was induced by treatment of IMR90 cells with doxo as judged by IP for pRB and WB for pRB and E2F1. (B–I) IMR90 cells were retrovirally transduced with either pBabe-hygro vector (−E1A) or pBabe-hygro-E1A12S (+E1A), selected with hygromycin 75μg/ml for four days and then assayed as follows. (B) The cells were treated with 2μM doxo (16 hours) and WB was used to determine the levels of pRB, E2F1, E2F3A, E2F4 and E1A in (left panel) pRB IPs and (right panel) the total lysates. The far right panel shows a longer exposure of the key input lanes. The E1A expression strongly increased both the total levels of pRB, E2F1 and E2F3A and the level of pRB-E2F1/E2F3A complexes. At the exposure shown, the pRB-E2F1 interaction is not visible in the −EIA;+doxo cells. (C) The binding of E1A to pRB and E2F1 is also detected via IP of E1A. (D) IPs with antibodies against ppRB and WB for pRB and E2F1. (E) The levels of pRB, E1A, E2F1 and AcH4 associated with the p73 and the CyclinA2 gene promoters were determined by ChIP. Densitometric quantification of each signal, relative to the input, is shown. (F) Real time RT-PCR analysis of Caspase 7, p73 and CyclinA2 mRNA levels. Results are expressed as arbitrary units normalized with GAPDH and they show the mean of three independent experiments (± SD). (G–I) Control or E1A-transduced cells were infected with either GFP or GFPshRB lentiviruses. (G) WB confirmed a reduction in pRB levels after shRB infection, using actin as the loading control. (H) Caspase 7, p73 and CyclinA2 mRNA levels were determined by real time RT-PCR analysis in cells cultured in the absence (−) or presence of 1μM etoposide (et) for 12 hours. Results are normalized with GAPDH and expressed relative to levels seen in the untreated cells (set as 1). Bars represent the mean of three independent experiments (± SD). (I) FACS analysis of early apoptotic cells (AnnexinV+, 7AAD−) after culture in the absence (−) or presence of 1μM et for 16 hours. Values are the percentage of apoptotic cells over the GFP+ cells (set at 1) and they represent the mean of three independent experiments (±SD).

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