Figure 1
Figure 1. Delayed maturation and altered Bcl-xL and Bim expression in Stat5−/− fetal liver. (A) Representative flow cytometric CD71/Ter119 fluorescence profiles of Stat5−/− fetal livers and wild-type littermates freshly isolated on consecutive embryonic days (E11.5 to E14.5). S0 to S4/5 are increasingly differentiated erythroid progenitors and precursors subsets in the fetal liver.36 Dead cells were excluded using LIVE/DEAD viability dye. (B) Summary of analysis performed as in panel A. Data points are each mean ± SEM of 4 to 21 embryos. Statistically significant differences between wild-type and Stat5−/− subsets (★) were found for S1 on E12.5 (P = .002, 2-tailed t test, unequal variance), E13.5 (P = .00001), and E14.5 (P = .014) and for S3 on E12.5 (P = .005), E13.5 (P = .00004), and E14.5 (P = .010). (C) Representative flow cytometry histograms for the Bcl-xL and Bim proteins in the indicated fetal liver subsets. Freshly isolated wild-type E14.5 fetal liver cells were stained with CD71, Ter119, and the LIVE/DEAD viability dye, and were then fixed, permeabilized, and stained intracellularly with an anti–Bcl-xL antiserum or nonimmune antiserum control (IgG), or with anti-Bim Ab or IgG isotype control. The x-axis is in fluorescence units. (D) Bcl-xL and Bim protein levels, measured as in panel C, in fresh wild-type fetal liver subsets S0 to S4/5 at the indicated embryonic days. Data were pooled from several experiments with multiple litters. Data points are each the median fluorescence intensity (MFI) ± SEM of 4 to 14 embryos or 6 embryos for Bcl-xL and Bim measurements, respectively. Nonspecific background fluorescence, defined as the MFI of the corresponding subset stained with control IgG, was subtracted. On E13.5 and E14.5, there were significant differences between S2 and S3 (P ≤ .0001), and between S3 and S4/5 (P < .001, paired t test). Statistically significant differences in Bim expression were found for S1 between E11.5 and E12.5 (P < .0001, 2-tailed t test, unequal variance). On E12.5, there were significant differences between S0 and S1 (P < .0001), and between S1 and S3 (P < .0001). Similar developmental patterns were observed in C57BL/6 and Balb/C backgrounds. (E) Lower Bcl-xL and higher Bim levels in E14.5 Stat5−/− embryos compared with wild-type littermate controls, at the indicated differentiation subset. For Bcl-xL, n = 11 to 21 embryos per genotype, with each symbol type representing median expression for 1 litter. Means ± SEM for the population are indicated. Statistically significant differences were found for S2 (P = .03), S3 (P = .002, paired t test). For Bim, data points are individual embryos. Mean ± SEM for the population is shown. Statistically significant differences were found for S3 and S4-5 (P < .01, 2-tailed t test, unequal variance).

Delayed maturation and altered Bcl-xL and Bim expression in Stat5−/− fetal liver. (A) Representative flow cytometric CD71/Ter119 fluorescence profiles of Stat5−/− fetal livers and wild-type littermates freshly isolated on consecutive embryonic days (E11.5 to E14.5). S0 to S4/5 are increasingly differentiated erythroid progenitors and precursors subsets in the fetal liver.36  Dead cells were excluded using LIVE/DEAD viability dye. (B) Summary of analysis performed as in panel A. Data points are each mean ± SEM of 4 to 21 embryos. Statistically significant differences between wild-type and Stat5−/− subsets (★) were found for S1 on E12.5 (P = .002, 2-tailed t test, unequal variance), E13.5 (P = .00001), and E14.5 (P = .014) and for S3 on E12.5 (P = .005), E13.5 (P = .00004), and E14.5 (P = .010). (C) Representative flow cytometry histograms for the Bcl-xL and Bim proteins in the indicated fetal liver subsets. Freshly isolated wild-type E14.5 fetal liver cells were stained with CD71, Ter119, and the LIVE/DEAD viability dye, and were then fixed, permeabilized, and stained intracellularly with an anti–Bcl-xL antiserum or nonimmune antiserum control (IgG), or with anti-Bim Ab or IgG isotype control. The x-axis is in fluorescence units. (D) Bcl-xL and Bim protein levels, measured as in panel C, in fresh wild-type fetal liver subsets S0 to S4/5 at the indicated embryonic days. Data were pooled from several experiments with multiple litters. Data points are each the median fluorescence intensity (MFI) ± SEM of 4 to 14 embryos or 6 embryos for Bcl-xL and Bim measurements, respectively. Nonspecific background fluorescence, defined as the MFI of the corresponding subset stained with control IgG, was subtracted. On E13.5 and E14.5, there were significant differences between S2 and S3 (P ≤ .0001), and between S3 and S4/5 (P < .001, paired t test). Statistically significant differences in Bim expression were found for S1 between E11.5 and E12.5 (P < .0001, 2-tailed t test, unequal variance). On E12.5, there were significant differences between S0 and S1 (P < .0001), and between S1 and S3 (P < .0001). Similar developmental patterns were observed in C57BL/6 and Balb/C backgrounds. (E) Lower Bcl-xL and higher Bim levels in E14.5 Stat5−/− embryos compared with wild-type littermate controls, at the indicated differentiation subset. For Bcl-xL, n = 11 to 21 embryos per genotype, with each symbol type representing median expression for 1 litter. Means ± SEM for the population are indicated. Statistically significant differences were found for S2 (P = .03), S3 (P = .002, paired t test). For Bim, data points are individual embryos. Mean ± SEM for the population is shown. Statistically significant differences were found for S3 and S4-5 (P < .01, 2-tailed t test, unequal variance).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal