Journal: Nature Communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression

doi: 10.1038/ncomms10028

Figure Lengend Snippet: ( a ) E2f1, E2f2, E2f3, E2f4 and Gapdh (normalization) expression levels in control liver and TKO HCC, as detected by immunoblotting. ( b ) IP for HA, E2f1 and E2f3 in primary liver extracts from cTKO (d0), Rosa26-CreER T2 TKO 4 days after Tamoxifen treatment (d4) and TKO HCC. Reptin (upper panels) and Pontin (middle panels) were detected in the pull-down fractions at each time point by immunoblotting. Expression levels of E2f1, E2f3, Reptin and Pontin were detected at the different time points in the corresponding input fractions by immunoblotting (lower panels). ( c ) H2a.z, Pontin, Reptin and Actin protein expression in control liver and TKO HCC, as detected by immunoblotting. Ponceau staining is used for normalization purpose. ( d ) mRNA expression for Reptin and Pontin in control liver ( n =5) and TKO HCC ( n =9), as detected by qPCR. ( e ) mRNA expression for H2a.z in control liver ( n =5) and TKO HCC ( n =9), as detected by qPCR. ( f ) Binding of E2f1 to the promoter region of H2a.z (following the identification of an E2f-binding site in the promoter of H2a.z ), as shown by ChIP assay in TKO HCC cells ( n =3). ( g ) Repression of H2a.z expression in two clones of TKO HCC cells (2.1 and 1.1) upon infection with retroviruses either control (MigR1) or expressing the Rb-7LP protein ( n =3). ( h ) Left: Frequency of E2F1 expression in tissue microarray (TMA) encompassing human HCC cases from stages I–IV. Right: Representative E2F1 staining of two independent HCC cases classified as stage IV HCC. Error bars represent standard deviation * P <0.05; ** P <0.01; *** P <0.001. WB, western blot.

Article Snippet: Antibody used: E2F1 (sc-193, 1/1,000), E2F2 (sc-833, 1/1,000), E2F3 (sc-893, 1/1,000), E2F4 (sc-1082, 1/500)), Actin (Cell Signaling, 4970, 1/10,000), GAPDH (sc-25778, 1/5,000), Pontin (Sigma, SAB4200194, 1/500), Reptin (Sigma, SAB1200115, 1/1,000), H2a.z (ab4174, 1/1,000), Glut4 (ab33780, 1/1,000), Gsk3b (Cell Signaling, 56765, 1/1,000).

Techniques: Expressing, Control, Western Blot, Staining, Binding Assay, Clone Assay, Infection, Microarray, Standard Deviation