Fig 2: Establishment of SRC-3-overexpressing MCF-7 cells. (A) Immunoblot analysis showing SRC-3 protein expression in MCF-7 cells transfected with pCMX-RAC3 (SRC-3) or pcDNA3 and selected for puromycin resistance. SRC-3 stable cells were categorized according to the relative size of the average cell within a colony prior to expansion. Examples of clones that survived at least one passage are shown: control pcDNA3 cells (1,2,3), small-sized SRC-3 transfected clones (S1,2,5) and medium-sized SRC-3 clones (M1,2,4). Large cell clones did not survive passage. (B) Immunoblot of original clones showing SRC-3 expression relative to empty vector-transfected control. (C) Flow analysis of DNA content derived from the indicated clones. (D) DNA content graphs derived from the analysis of several clones. Bars are SD of means from: pcDNA control (n = 4), SRC-3 small (n = 15), SRC-3 medium (n = 4). (E) Phase-contrast images of control pcDNA3- and pCMX-RAC3- (SRC-3) transfected MCF-7 cells. Panels are (i) Control clones, (ii) small SRC-3-overexpressing clones, (iii and iv) medium SRC-3-overexpressing clones which were enlarged and flat with abundant cytoplasm. (F) Cell lysates harvested after infection at the indicated times after infection with Ad-LacZ or Ad-SRC-3 were probed with antibodies to SRC-3, P-Chk2, Chk2 (denoted by arrowhead), p21 and actin. (G) MCF-7 cells infected with the Ad-RFP and Ad-SRC-3 viruses were cultured for 72 h and assayed for senescence-associated (SA) β-galactosidase activity. (H) Immunoblot for SRC-3, cyclin E and PARP-1 in MCF-7 cell lysates 72 h post-transfection with empty vector (EV), wtSRC-3 or the stable mutant SRC-3(S102A). Note that although highly expressed relative to wtSRC-3 and to the gel loading control, S102A does not induce transcription of cyclin E. Actin was used as a protein loading control.

Fig 3: Effect of β-catenin nuclear translocation on cell cycle distribution in the prostate cancer cell lines, LNCaP and C4-2B. (A) Western blot analysis of the protein expression of cell cycle regulatory proteins, including p21, CDK1, p-CDK1, Chk1, p-Chk1, Chk2, p-Chk2, Rb and p-Rb at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells. (B–F) Statistic analysis of the expression of cell cycle regulators. Bar 1 indicates untreated cells, bar 2 indicates PcDNA3.1(−)/HIF-1α plasmid transfected cells, and bar 3 indicates HIF-1α high expression cells transfected with PSuper-β-catenin-shRNA plasmid. ●P>0.05; ▲P<0.05; *P<0.01; **P<0.001.

Fig 4: HBX specifically inhibits CHK2 phosphorylationa, b Western blotting for γH2AX in control and HBX-expressing SUDHL-4 a, and DB b, cells with or without MTX treatment (4 ng/ml) for 48 h. c, d Western blotting for DDR proteins (P-CHK2/CHK2/ATM and P-CHK1/CHK1) in control and HBX-expressing SUDHL-4 c, and DB d, cells with or without MTX treatment (4 ng/ml) for 48 h. e, f The mRNA levels of DDR proteins (ATM, CHK2, and CHK1) were largely unchanged in control and HBX-expressing SUDHL-4 e, and DB f, cells with or without MTX treatment. g, h The expression of the P-CHK2 downstream genes P53 and P21 in control and HBX-expressing SUDHL-4 g, and DB h, cells with or without MTX treatment. The results are shown as the mean ± SEM from triplicate experiments. GAPDH was used as a loading control

Fig 5: HPV16 E6 ability to induce p53 degradation is critical to sensitize primary human keratinocytes to ATM and CHEK2 silencing or kinase activity inhibition. PHK were transduced with retroviral vectors expressing wild type HPV-16 E6 and/or E7, or an HPV-16 mutant E6 unable to induce p53 degradation (16ΔE6). The expression of (A) ATM and (B) CHEK2 was silenced using specific shRNAs. Cells were seeded in 96-wells plates (5000 cells/well) and after 72 h their viability was assessed by Alamar Blue reduction. The activity of ATM and CHK2 kinases was inhibited using (C) caffeine (an inhibitor of ATM and ATR), (D) a specific inhibitor ATM and (E) a specific inhibitor of CHK2. Cells were seeded in 96-wells plates (5000 cells/well). Cells were treated with the inhibitors for 72 h and their viability was assessed by Alamar Blue reduction. The proliferation of PHK expressing HPV-16 E6 and transduced with a shRNA against the ubiquitin ligase responsible for p53 degradation in the presence of E6 (E6-AP) was not strongly affected by ATM or CHK2 inhibition (C–E). Results presented are representative of three independent experiments (Student t-test p < 0.05 [*] for all comparisons between control and treated cells).