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. 2014 Apr 14;5:3649. doi: 10.1038/ncomms4649

Figure 5. PRC1 is required for proliferation and transformation independently of the Ink4a/Arf-p53-pRb axis.

Figure 5

(a) Growth curves, immunoblots and BrdU FACS analysis of Ring1a −/−, Ring1b fx/fx and Ring1a −/− and Ring1b −/− MEFs grown at 3% O2 for the indicated times. Left panels show the crystal violet staining of cells at day 5 of the growth curve. Quantification of crystal violet absorbance at λ=590 nm at the indicated time points is depicted in the graph. Middle panel shows immunoblots using the indicated antibodies of protein extracts derived from Ring1a −/− Ring1b fx/fx and Ring1a −/−, Ring1b −/− MEFs at day 5. β-tubulin and total histone H2A served as loading controls. Right panel shows percentage of BrdU incorporation measured by FACS analysis between Ring1a −/−, Ring1b fx/fx and Ring1a −/− and Ring1b −/− MEFs. In all growth curves, error bars indicate s.d., n=3. (b) Growth curves and immunoblots as in a, using Ink4a/Arf −/−, Ring1a −/−, Ring1b fx/fx or Ring1b −/− MEFs at 3% O2. β-tubulin and total histone H2A served as loading controls. (c) Growth curves as in a with SV40ER-immortalized Ring1a −/− or Ring1b fx/fx MEFs treated with ethanol (Ring1b fx/fx) or OHT (Ring1b −/−) at 3% O2. (d) Immunoblots were performed with the indicated antibodies using protein extracts prepared from SV40ER-immortalized Ring1a −/− or Ring1b fx/fx MEFs in which OHT treatment and H-RASV12 expression was combined with the order indicated in the figure. β-tubulin and total histone H2A served as loading controls. (e,f) Tumour formation of the cells described in d by injection in both flanks of athymic nude mice. Pictures of mice and of the isolated tumour masses at 14 days post injection are shown in e. The average increase of tumour size at the indicated time (left) and the weights of the tumour masses shown in e are presented in f. Error bars indicate s.d., n=10. P-value was determined using a Mann–Whitney test. Scale bar, 1 cm.