Figure 6
Figure 6. Adaptation in the Bcl-xL and p-Stat5 responses is dependent on the EpoR C-terminal cytoplasmic domain. (A) The p-Stat5 response to Epo in vivo in freshly explanted spleen ProE and EryA at the indicated times after a single Epo injection (300 U/25 g). Data were pooled from 4 independent experiments. Each time point is the mean ± SEM of data from 2 to 4 mice. (B) The p-Stat5 time course in S1 fetal liver cells in response to Epo stimulation (2 U/mL), shown for EpoR-HM, EpoR-H, and matched wild-type fetal livers at E13.5. Data are MFI above background (isotype-control Ab). Representative of 3 similar experiments. (C) The Bcl-xL response to Epo in vivo in EpoR-H and EpoR-HM mice. Bcl-xL was measured 18 hours after a single injection of either saline or Epo (300 U/25 g), in freshly explanted spleen ProE and EryA of EpoR-H, EpoR-HM, or wild-type controls. Data are mean ± SEM of n = 3 to 5 mice per bar. Significant Bcl-xL increase from basal levels in spleen ProE and EryA was seen in Epo vs saline-injected wild-type (black) and EpoR-H (red) mice (stars without brackets: WT ProE *P = .003; EpoR-H ProE *P = .012; WT EryA *P = .00004; EpoR-H EryA *P = .0001, 2-tailed t test, unequal variance), but not in EpoR-HM mice (blue). Bcl-xL was reduced in basal state EpoR-HM spleen EryA (red star with red bracket, *P = .027) compared with wild-type basal control. Bcl-xL induction in wild-type spleen EryA was significantly above that of EpoR-HM EryA (black star with bracket, *P = .007). (D) Time course of the Bcl-xL response in EpoR-H mice and in matched wild-type controls, after a single Epo injection (300 U/25 g). Measurements were made in freshly explanted spleen at the indicated time points. Bcl-xL is significantly higher in EpoR-H at 36 and 48 hours (P < .005, paired t test on all subsets). (E) Bim protein in spleen ProE and EryA of wild-type and EpoR-HM mice on day 3 after a single Epo injection (300 U/25 g). Data are mean ± SEM of n = 4 to 5 mice per bar. There was no significant difference in basal Bim between EpoR-HM and wild-type control mice. Bim was significantly suppressed after Epo injection (*P < .001). Bim was suppressed by a significantly smaller extent in EpoR-HM ProE and EryA subsets (stars with brackets, *P = .03 and *P = .001, respectively, 2-tailed t test, unequal variance). (F) The p-Stat5 histograms in vivo at peak response (30 minutes) after a single injection of either Epo (300 U/25 g) or saline, in either β-thalassemia mice or in matched wild-type controls, measured in freshly explanted spleen ProE. The p-Stat5+ gate was drawn based on the nonerythroid population in spleen (gray histograms). The x-axis is in fluorescence units.

Adaptation in the Bcl-xL and p-Stat5 responses is dependent on the EpoR C-terminal cytoplasmic domain. (A) The p-Stat5 response to Epo in vivo in freshly explanted spleen ProE and EryA at the indicated times after a single Epo injection (300 U/25 g). Data were pooled from 4 independent experiments. Each time point is the mean ± SEM of data from 2 to 4 mice. (B) The p-Stat5 time course in S1 fetal liver cells in response to Epo stimulation (2 U/mL), shown for EpoR-HM, EpoR-H, and matched wild-type fetal livers at E13.5. Data are MFI above background (isotype-control Ab). Representative of 3 similar experiments. (C) The Bcl-xL response to Epo in vivo in EpoR-H and EpoR-HM mice. Bcl-xL was measured 18 hours after a single injection of either saline or Epo (300 U/25 g), in freshly explanted spleen ProE and EryA of EpoR-H, EpoR-HM, or wild-type controls. Data are mean ± SEM of n = 3 to 5 mice per bar. Significant Bcl-xL increase from basal levels in spleen ProE and EryA was seen in Epo vs saline-injected wild-type (black) and EpoR-H (red) mice (stars without brackets: WT ProE *P = .003; EpoR-H ProE *P = .012; WT EryA *P = .00004; EpoR-H EryA *P = .0001, 2-tailed t test, unequal variance), but not in EpoR-HM mice (blue). Bcl-xL was reduced in basal state EpoR-HM spleen EryA (red star with red bracket, *P = .027) compared with wild-type basal control. Bcl-xL induction in wild-type spleen EryA was significantly above that of EpoR-HM EryA (black star with bracket, *P = .007). (D) Time course of the Bcl-xL response in EpoR-H mice and in matched wild-type controls, after a single Epo injection (300 U/25 g). Measurements were made in freshly explanted spleen at the indicated time points. Bcl-xL is significantly higher in EpoR-H at 36 and 48 hours (P < .005, paired t test on all subsets). (E) Bim protein in spleen ProE and EryA of wild-type and EpoR-HM mice on day 3 after a single Epo injection (300 U/25 g). Data are mean ± SEM of n = 4 to 5 mice per bar. There was no significant difference in basal Bim between EpoR-HM and wild-type control mice. Bim was significantly suppressed after Epo injection (*P < .001). Bim was suppressed by a significantly smaller extent in EpoR-HM ProE and EryA subsets (stars with brackets, *P = .03 and *P = .001, respectively, 2-tailed t test, unequal variance). (F) The p-Stat5 histograms in vivo at peak response (30 minutes) after a single injection of either Epo (300 U/25 g) or saline, in either β-thalassemia mice or in matched wild-type controls, measured in freshly explanted spleen ProE. The p-Stat5+ gate was drawn based on the nonerythroid population in spleen (gray histograms). The x-axis is in fluorescence units.

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