Fig 1: Senescence growth arrest in CL3EcoR cells. (a) Senescence specific changes in expression at the RNA level as determined by expression profiling are shown. GA indicates Log2 fold changes in RNA expression upon senescence growth arrest. Positive numbers for GA indicate up-regulation and are shaded in red, (log2 fold change less than 0.5 are shaded in light red). Negative numbers indicating down-regulation are shaded in green (log2 fold change less than − 0.5 are shaded in light green). Fold changes in expression of LIN9 and RBL1 are shown for 2 and 3 independent features whereas all the others are for a single feature only. Also shown is the effect on expression when senescence was bypassed by inactivation of the pRB/p16INK4A and p53/p21WAF1/CIP1 pathways7. The pRB/p16INK4A pathway was inactivated using SV40 wild type LT (wt_LT), Adenovirus 5 E1A 12S (E1A), Human Papilloma Virus (HPV) 16 E7 (E7) or a dominant negative E2F-DB protein7,21. The p53/p21WAF1/CIP1 pathway was inactivated using SV40 wild type LT, a p53GSE element that inactivates p53 (GSE-p53)22 or short-hair pin RNAs targetting p53 (pRS_p53)23 or p21WAF1/CIP1 (pRS_p21)7. The changes in expression were reversed upon senescence bypass. (b) Representative immunoblots assessing changes in protein levels of FOXM1, B-MYB, LIN9, LIN37, LIN52, LIN54, DYRK1A, RB1, pRBS780, p107, and p130 in CL3EcoR and HMF3S cells. Images of uncropped gels and blots are shown in the Supplementary Figures.
Fig 2: Senescence bypass assays using individual components of the MMB-FOXM1 complex. (a) Computational analysis of single representative flasks upon ectopic expression of the indicated component is shown. Blue spots depict the area of the flask computed as blue by a computer algorithm. Quantitative representation of the bypass assay is presented graphically by plotting the calculated percentage area (+ /− SD) covered in blue by Definiens Developer XD software for each of the replicate flasks. Constitutively active LIN52 (LIN52-S28A) and FOXM1 (FOXM1ΔNΔKEN) were utilised. The strongest bypass was exhibited by B-MYB and LIN52-S28A. For a quantitative analysis, the bar chart depicts an overall average (+ /− SD) of the two independent repeat experiments. Empty pLEX-MCS vector was used as the negative control. Statistical analysis was conducted using One-way ANOVA, Tukey’s Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (b) Comparisons of the senescence bypass potential of wild type LIN52 and mutants thereof. LIN52 was inactive whereas both LIN52 mutants (LIN52-S28A and LIN52-S28E) exhibited senescence bypass activity. For quantitative analysis the bar charts depict an overall average (+ /− SD) of the three independent repeat experiments. Empty pLEX-MCS vector was used as negative control. Statistical analysis was conducted using One-way ANOVA, Tukey’s Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Fig 3: Non-phosphorylated LIN52, FOXM1 and B-MYB are critical for bypassing senescence. (a) Bypass assays of the reconstituted MMB-FOXM1 complex lacking one component. The highest numbers of densely growing colonies were observed for the whole reconstituted complex “RC” and “X LIN54” in which LIN54 had been omitted. “X LIN9” and “X LIN37” produced the next highest whereas “X LIN52-S28A”, “X FOXM1ΔNΔKEN” and “X B-MYB” produced the least of which “X LIN52-S28A” lacking LIN52-S28A was the lowest. For qualitative analysis single representative flasks from internal repeats are shown with the component absent from the reconstituted complex as indicated. “X” indicated in front of a component depicts its absence in the reconstituted complex. For quantitative analysis, the bar charts depict an overall average (+ /− SD) of three-independent repeat experiments. Empty pLEX-MCS vector and RAD51 were used as the negative control. Statistical analysis was conducted using One-way ANOVA, Tukey’s Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (b) LIN52, FOXM1 and B-MYB together synergize to bypass senescence. A significant difference in the bypass potential of the reconstituted MMB-FOXM1 complex was observed with respect to each of the critical components. For quantitative analysis the bar charts depict an overall average (+ /− SD) of three-independent repeat experiments. Empty pLEX-MCS vector was used as a negative control. Statistical analysis was conducted using One-way ANOVA, Tukey’s Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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