mRNA levels of and in passage 3 MEFs (Figure 4), and and Ink4a, Arf p21 Ink4a Arf levels in passage 5 MEFs (Figure 1e). n = 4 independent cultures of each genotype. Actin is provided as a loading control. Supplementary Figure 7 Expression analysis of wild type and MEFs. Moz-/- – (a) mRNA was not detected in MEFs (b) Gene expression levels of major Moz Moz-/- regulators of senescence in wild type and MEFs grown at physiological 3% Moz-/- oxygen. mRNA levels of genes encoding major inhibitors of senescence that were decreased in MEFs at 20% O2 (Fig. 5) were also decreased at 3% O2 (here). Moz-/- This suggests that the reduction in mRNA levels of and Cdc6, Skp2, Ezh2, Melk, E2f2 was independent of DNA damage, which is prevalent in MEFs cultured in 20% Nsd2 O2. (c) mRNA levels in cells were not different to Ink4b Ink4a-Arf-/-;Moz-/- controls (d) Ratio of housekeeping genes mRNA to mRNA were Hsp90ab1 Gapdh not different between wild type and cells at 20% O2. n = 4 independent cultures Moz-/- of each genotype. Gene expression levels were standardised to housekeeping genes and Asterisks mark statistically significant differences between wild Hsp90ab1 Gapdh. type and cultures at *p < 0.05, **p < 0.01 and ***p < 0.001. Moz-/- Supplementary Figure 8 CDC6 overexpression affords a modest growth advantage – to MEFs. MEFs were virally infected with a vector and control Moz-/- Cdc6-GFP negative vector. Infection with a vector lead to a small but Empty-GFP Cdc6-GFP statistically significant increase in cells compared to controls. n = 3 independent Moz-/- cultures of each genotype. Asterisks mark statistically significant differences between wild type and cultures at *p < 0.05. Moz-/-
mRNA levels of Ink4a, Arf and p21 in passage 3 MEFs (Figure 4), and Ink4a and Arf levels in passage 5 MEFs (Figure 1e). n = 4 independent cultures of each genotype. Actin is provided as a loading control. Supplementary Figure 7 – Expression analysis of wild type and Moz-/- MEFs. (a) Moz mRNA was not detected in Moz-/- MEFs (b) Gene expression levels of major regulators of senescence in wild type and Moz-/- MEFs grown at physiological 3% oxygen. mRNA levels of genes encoding major inhibitors of senescence that were decreased in Moz-/- MEFs at 20% O2 (Fig. 5) were also decreased at 3% O2 (here). This suggests that the reduction in mRNA levels of Cdc6, Skp2, Ezh2, Melk, E2f2 and Nsd2 was independent of DNA damage, which is prevalent in MEFs cultured in 20% O2. (c) Ink4b mRNA levels in Ink4a-Arf-/-;Moz-/- cells were not different to controls (d) Ratio of housekeeping genes Hsp90ab1 mRNA to Gapdh mRNA were not different between wild type and Moz-/- cells at 20% O2. n = 4 independent cultures of each genotype. Gene expression levels were standardised to housekeeping genes Hsp90ab1 and Gapdh. Asterisks mark statistically significant differences between wild type and Moz-/- cultures at *p < 0.05, **p < 0.01 and ***p < 0.001. Supplementary Figure 8 – CDC6 overexpression affords a modest growth advantage to Moz-/- MEFs. MEFs were virally infected with a Cdc6-GFP vector and control negative Empty-GFP vector. Infection with a Cdc6-GFP vector lead to a small but statistically significant increase in Moz-/- cells compared to controls. n = 3 independent cultures of each genotype. Asterisks mark statistically significant differences between wild type and Moz-/- cultures at *p < 0.05.
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