1. Transcriptional Regulation of AKT Activation by E2F
Marie Chaussepied, Doron Ginsberg 
Molecular Cell 
Volume 16, Issue 5, Pages (December 2004)
DOI: /j.molcel

2. Figure 1 E2F Transcriptionally Regulates Phospho-AKT Levels
(A) U-2OS cells expressing nonspecific shRNA (NS) or shRNA specific for E2F1 and E2F3 that had been synchronized in mitosis (0h) were allowed to resume cell cycle progression. Proteins were extracted from mitotic cells and cells at different time after release into the cell cycle. Phospho-AKT (p-AKT), AKT1, E2F1, and E2F3 levels were analyzed by Western blot (lower panel). The corresponding cell cycle distribution is shown (upper panel).
(B) U-2OS cells were infected with an empty retrovirus (−), a retrovirus expressing ER-E2F1 wild-type (wt), or a retrovirus expressing ER-E2F1-E132 (E132). Infected cells were serum starved and then left untreated (−) or treated with OHT for 8 hr (+). Phospho-AKT (p-AKT), AKT1, phospho-GSK-3α/β (p-GSK3), and GSK-3β levels were analyzed by Western blot.
(C) Serum-starved U-2OS cells expressing ER-E2F1 were treated with OHT for the indicated duration. Where indicated, thymidine was added 16 hr prior to OHT addition. Phospho-AKT (p-AKT) and AKT1 levels were analyzed by Western blot. For each time point, the percentage of BrdU-positive cells is shown.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Gab2 Is a Direct Transcriptional Target of E2F
(A) RT-PCR analysis of Gab2 and GAPD mRNA levels in serum-starved, parental, and ER-E2F1-expressing U-2OS cells treated for 4 hr with OHT (+) or not treated (−).
(B) RT-PCR analysis of Gab2 and GAPD mRNA levels in serum-starved, ER-E2F1-expressing U-2OS cells treated with OHT for 4 hr (+) or not treated (−) in the presence or absence of cycloheximide (CHX).
(C) ChIP analysis was performed using growing Jurkat cells: Crosslinked chromatin was immunoprecipitated with antibodies to E2F1, E2F2, E2F3, E2F4, and HA or without antibody (−), and then Gab2 and β-actin (ACTB) promoter fragments were amplified by PCR. Negative (no DNA) and positive (input DNA representing 0.2% of total input chromatin) control amplifications are shown.
(D) U-2OS cells were transiently cotransfected with the −477/+88-Gab2-luciferase construct and CMVßGal plasmid together with either empty vector, E2F1 E132 expression vector (50 ng), or increasing amounts of wt E2F1 expression vector (50, 100, and 200 ng). Luciferase and β-galactosidase activities were measured 24 hr after transfection. Results are depicted as fold induction, after normalization for β-Gal activity, compared to U-2OS cells transfected with pGL3-basic (value set at 1), and represent the average of three independent experiments performed in duplicate. The bottom panel shows a Western blot analysis of E2F1 expression levels in a representative transfection experiment.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 E2F Activity Increases Gab2 mRNA and Protein Levels
(A) RT-PCR analysis of Gab2, Cyclin E1 (CCNE1), and a control (GAPD) mRNA levels in Bcl-xL expressing U-2OS cells infected with a control retrovirus (−) or retroviruses expressing either wt E2F1 or E2F1 E132.
(B) Western blot analysis of Gab2 and GAPD (as a loading control) protein levels in Bcl-xL expressing U-2OS cells infected with an empty retrovirus (−), a retrovirus expressing wild-type E2F1 (wt), or a retrovirus expressing E2F1-E132 (E132).
(C) RT-PCR analysis of Gab2, Cyclin E1 (CCNE1), and control (EEF1α1) mRNA levels in Bcl-xL expressing WI38 cells infected with control retrovirus (−) or a retrovirus expressing E1A (+).
(D) U-2OS cells synchronized in mitosis by nocodazole treatment (0h) were allowed to resume cell cycle progression. RNA and proteins were extracted from mitotic U-2OS cells and U-2OS cells at different time after release into the cell cycle. Gab2 and Cyclin E1 (CCNE1) relative mRNA levels were determined by real-time RT-PCR and normalized to HPRT relative levels. Gab2 and vinculin (as a loading control) protein levels were analyzed by Western Blot (middle panel). For each time point, cell cycle distribution and percentage of cells with a sub-G1 DNA content are indicated (lower panel).
(E) Serum-starved U-2OS cells expressing ER-E2F1 were treated with OHT for the indicated duration. Where indicated, thymidine was added 16 hr prior to OHT addition (for the corresponding percentage of cells in S phase, see Figure 1C). Gab2 relative mRNA levels were determined by real-time RT-PCR and normalized to β-2-microglobulin (b2m) relative levels. Gab2 and vinculin (as a loading control) protein levels were analyzed by Western Blot (lower panel). For each time point, the percentage of BrdU-positive cells was analyzed (see Figure 1C).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Gab2 Is Required for E2F1-Mediated AKT Activation
(A) U-2OS cells expressing ER-E2F1were infected with an empty retrovirus (−), a retrovirus expressing wild-type Gab2 (wt), or a retrovirus expressing Gab2 3YF (3YF). Infected cells were serum starved for 48 hr and then either left untreated (−) or treated with OHT for 8 hr (+). Phospho-AKT (p-AKT) and AKT1 levels were analyzed by Western blot.
(B) U-2OS cells expressing ER-E2F1 were infected with pRETRO-SUPER retroviruses expressing either nonspecific shRNA (NS1 and NS2) or Gab2-specific shRNA (Gab21 and Gab22). Infected cells were serum starved for 48 hr and then either left untreated (−) or treated with OHT for 4 hr (+). Gab2-relative mRNA levels were determined by real-time RT-PCR and normalized to β-2-microglobulin relative mRNA levels.
(C) Extracts from the U-2OS cells described in (B) were used for Western blot analysis of Gab2, vinculin, phospho-AKT (p-AKT), and AKT1 protein levels. Cells were serum starved for 48 hr and then either left untreated (−) or treated with OHT for 8 hr (+).
Molecular Cell  , DOI: ( /j.molcel )