Fig 2: Therapy-induced senescence reduces readthrough in cancer cells. (A) SA-ß-gal staining of PC-3 cells treated 24 h with vehicle and/or 100 nM of camptothecin and fixed 7 days later (day 7), the percent positive cells ±SD is shown in lower corner, n=3. (B) Immunoblots in PC-3 cells transduced with luciferase reporter (AQP4, UGA) and treated as in A for the proteins: RB(pS795) [phosphorylated RB on serine 795], MCM6, H3(pS10) [phosphorylated H3 on serine 10] and tubulin. Blots are representative of three independent experiments with similar results. (C) Normalized Fluc/Rluc ratios of indicated readthrough reporters in cells as in A. Normalizations are presented as means relative to vehicle treated cells from (UGA) n=4 and (AQP4) n=5 independent experiments each with technical triplicates. (D) Normalized Fluc/Rluc ratios in of MDA.MB.231 cells transduced with the luciferase reporters (UGA, AQP4) and treated as in A. Normalizations are presented as means relative to vehicle treated cells, n=3. (E) Immunoblots for phosphorylated RB at serine 795 [RB(pS795)], MCM6 and tubulin in cells as in D, n=3. (F) Normalized Fluc/Rluc ratios of indicated readthrough reporters in PC-3 cells as in A, 21 days post-treatment when the cell population bypassed senescence. For all luciferase assays, n=3, error bars indicate SD of technical triplicates of each experiment. *=P<0.05, **=P<0.01, ***=P<0.001 using two-tailed Student's t-test. (G) Immunoblots for phosphorylated RB at serine 795 [RB(pS795)] and tubulin in cells as in E, n=3.

Fig 3: The regulation of p53 target genes by SOCS1 is not dependent on a disabled RB pathway(A) QPCR validation of the p53 target genes identified by microarray analysis but in normal IMR90 fibroblasts expressing either an empty vector (V) or a constitutively activated STAT5A (cS5A) and with either a control shRNA (shNTC) or an shRNA against SOCS1 (shS1). Cells were collected 7 days after infection. (B) SOCS1 knockdown efficiency measured by qPCR in the conditions described in (A). (C) Status of the cells was assessed at the day of RNA collection (day 7 post infection) with a Senescence-Associated ß-Galactosidase staining. Positively stained and unstained cells were counted under a light microscope in order to obtain the percentage of senescent cells. (D) Growth curves. Normal human fibroblasts (IMR90) were retrovirally infected with either an empty vector (V) or with constitutively activated STAT5A (cS5A) and with either a control shRNA (shNTC) or a shRNA against SOCS1 (shS1 a). Cells were counted and plated for the growth assay. (E) Western blots of senescence markers (MCM6, pRb and SOCS1) of cells as in (A). Tubulin was used as a loading control. All experiments were performed three times, error bars indicate the standard errors of triplicates, * = p<0.05, using the Student's t test, **=p<0.01, ***=p<0.005

Fig 4: HsGeminin inhibits the ATP-dependent salt-stable binding of HsMCM2–7 to DNA. A, requirements for formation of salt-stable association of HsMCM2–7 with DNA. Reaction mixtures were assembled and incubated as described in the legend to Fig. 3B. The beads were then washed with buffer containing 0.3 m NaCl and bound proteins were analyzed by Western blotting. B, HsGeminin inhibits formation, but not the maintenance of salt-stable HsMCM2–7·DNA complexes. Reaction mixtures were assembled and incubated as described in the legend to Fig. 4C, except that HsMCM2–7 was used instead of HsMCM467, and HsCdc6 was also present. The beads were then washed with buffer containing 0.5 m NaCl. Lane 1: control buffer was added at t = -30 min. Lane 2: HsGeminin was added at t = -30 min. Lane 3: HsGeminin was added at t = 0 min. Lane 4: control buffer was added at t = +20 min. Lane 5: HsGeminin was added at t = +20 min. C, ATP-dependent formation of salt-stable HsMCM2–7·DNA complexes. Reactions were performed in the presence of ATP, ATP?S, ADP, or control buffer as indicated. The beads were then washed with buffer containing 0.3 m NaCl. D, HsGeminin inhibition of salt-stable complex formation. Reaction mixtures were incubated in the presence or absence of ATP as indicated. HsCdt1 was preincubated with control buffer or HsGeminin. The beads were then washed with buffer containing 0.3 m NaCl. E, ATP requirement in two-step DNA binding assays. Magnetic beads containing pUC19-lamin-B2 plasmid DNA were incubated with HsORC for 30 min, and then further incubated with HsMCM2–7, HsCdt1, and HsCdc6 for 30 min. ATP or ATP?S was added in each step as indicated. The beads were then washed with buffer containing 0.1 m potassium glutamate or 0.3 m NaCl and analyzed by Western blotting. Mcm6 l, long exposure of Mcm6 signal. Mcm6 s, short exposure of Mcm6 signal. Gem, Geminin.

Fig 5: Interactions between individual human MCM subunits and HsCdt1 or HsCdc6, and effects of HsGeminin on the interactions between MCM subunits and HsCdt1. A–D, extracts of Sf9 cells expressing untagged human Mcm4 (A), Mcm5 (B), Mcm6 (C), or Mcm7 (D) were incubated at 4 °C for 3 h with extracts from control cells or extract from cells expressing FLAG-HsCdt1 or Myc-HsCdc6. For each Mcm subunit, an equal amount of protein was added in each reaction. The proteins precipitated by anti-FLAG- or anti-Myc-agarose beads were analyzed by Western blotting. L indicates 8.3% of the load. E–H, extracts of Sf9 cells expressing untagged human Mcm3 (G), Mcm5 (H), Mcm6 (E), or Mcm7 (F) were incubated at 4 °C for 3 h with extracts from control cells or extracts from cells expressing FLAG-HsCdt1 and/or FLAG-HsGeminin-His6. The proteins precipitated by anti-FLAG-agarose beads were analyzed by Western blotting. IP, immunoprecipitated.