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. 2016 Mar;186(3):701–715. doi: 10.1016/j.ajpath.2015.10.028

Figure 8.

Figure 8

FBP helps to set and stabilize MYC levels. A: Loss of FBP is associated with increased cell-to-cell variation in MYC levels. MEFs were recovered at E13.5 from embryos with the indicated genotypes, cultured, stained for MYC, and quantified as described in Materials and Methods. B and C: Loss of FBP is associated with increased variability in MYC set points between individual embryos. Individual embryos (E14.5) from the litters of two concurrently sacrificed Fubp1+/− dams that had been mated with an Fubp1+/− male were genotyped with head tissue, while MEFs were prepared and cultured and individual extract was analyzed to reveal variable MYC set points. C: Immunoblots were analyzed for MYC and actin (or other housekeeping proteins, tubulin, glyceraldehyde-3-phosphate dehydrogenase, or histone; not shown). MEFs recovered from a WT × WT litter were similarly prepared and analyzed and shown in B and C as a reference. WT extracts are indicated with dots; the black, blue, and open dots show results derived from a single litter of WT embryos; the blue and open dots show technical duplicates of a single extract; only the blue dot was included in the statistical analysis with the other single embryo MEFs. The red dots indicate WT-MEFs recovered from two Fubp1+/−× Fubp1+/− litters. B and C: the results of immunoblots of MEFs from individual Fubp1−/− embryos are indicated with red squares. The MEFs from three of the four Fubp1−/− embryos (red squares) are outside the range of all WT MEFs. The open red dots in C show immunoblots of extracts from heterozygous Fubp1+/− single embryo MEFs that were not included in this analysis. D and E: A comparison is shown of the MYC levels normalized with tubulin from a collection of MEFs derived from either WT or Fubp1−/− single embryos. Although the difference between the means of the WT and Fubp1−/− is not significant at the 95% CI, the F test is positive at P < 0.0003, indicating that the Fubp1−/− MYC levels are significantly more variable than the WT. E, embryonic day; FBP, far upstream element binding protein; KO, knockout; MEF, mouse embryonic fibroblast; WT, wild-type.