Figure 6.
Figure 6. Suppression of erythroblast Fas and FasL and decreased apoptosis following Epo injection. Wild-type Balb/C mice were injected with a single dose of Epo (50 mg/kg) or with an equal volume of saline, and followed as described in Figure 2. Freshly isolated spleen cells were analyzed for Fas and FasL cell-surface expression and mRNAs or for apoptotic markers. (A) Representative histograms of cell-surface Fas (left panels) and FasL (middle panels) expression or annexin V binding (right panels) in Ery.A, B, and C, 48 hours following injection of either saline (top panels) or Epo (bottom panels). The percentage of Ery.A positive for Fas, FasL or annexin V is indicated in red, and that of Ery.B in blue. (B) Time course of cell-surface Fas and FasL following Epo or saline injection. Results pooled from 5 experiments, normalized to initial Fas or FasL levels; mean ± SEM, n = 2 to 7 mice per time point. (C) Time course of annexin V binding (geometric mean fluorescence) in ProEs (top panel) and in Ery.A (bottom panel) following Epo administration. Results from duplicate mice are shown for each time point. (D) Changes in activated caspase 3 in splenic erythroblasts following Epo injection. Splenic cells were labeled for Ter119 and CD71, were fixed and permeabilized, and then labeled with an antibody specific for activated caspase 3. Results from duplicate mice are shown for each time point. (E) Time course of Fas, FasB, and FasL mRNA expression in splenic Ery.A following injection with Epo, measured using quantitative real-time PCR, using RNA from freshly sorted splenic Ery.A. Bottom panels show the ΔΔCT for the indicated mRNA between mice injected with Epo and those given saline injections, normalized to actin mRNA. Each data point is the mean ± SEM of triplicate measurements in a single experiment using 1 to 2 Epo-injected mice and 1 to 2 saline-injected mice. Two to 4 independent experiments were conducted per time point. Top panels show the mean fold change in mRNA at each time point, calculated as 2ΔΔCT, using the ΔΔCT values shown in the bottom panels. Each data point is the mean ± SEM for all the mice examined at each time point.

Suppression of erythroblast Fas and FasL and decreased apoptosis following Epo injection. Wild-type Balb/C mice were injected with a single dose of Epo (50 mg/kg) or with an equal volume of saline, and followed as described in Figure 2. Freshly isolated spleen cells were analyzed for Fas and FasL cell-surface expression and mRNAs or for apoptotic markers. (A) Representative histograms of cell-surface Fas (left panels) and FasL (middle panels) expression or annexin V binding (right panels) in Ery.A, B, and C, 48 hours following injection of either saline (top panels) or Epo (bottom panels). The percentage of Ery.A positive for Fas, FasL or annexin V is indicated in red, and that of Ery.B in blue. (B) Time course of cell-surface Fas and FasL following Epo or saline injection. Results pooled from 5 experiments, normalized to initial Fas or FasL levels; mean ± SEM, n = 2 to 7 mice per time point. (C) Time course of annexin V binding (geometric mean fluorescence) in ProEs (top panel) and in Ery.A (bottom panel) following Epo administration. Results from duplicate mice are shown for each time point. (D) Changes in activated caspase 3 in splenic erythroblasts following Epo injection. Splenic cells were labeled for Ter119 and CD71, were fixed and permeabilized, and then labeled with an antibody specific for activated caspase 3. Results from duplicate mice are shown for each time point. (E) Time course of Fas, FasB, and FasL mRNA expression in splenic Ery.A following injection with Epo, measured using quantitative real-time PCR, using RNA from freshly sorted splenic Ery.A. Bottom panels show the ΔΔCT for the indicated mRNA between mice injected with Epo and those given saline injections, normalized to actin mRNA. Each data point is the mean ± SEM of triplicate measurements in a single experiment using 1 to 2 Epo-injected mice and 1 to 2 saline-injected mice. Two to 4 independent experiments were conducted per time point. Top panels show the mean fold change in mRNA at each time point, calculated as 2ΔΔCT, using the ΔΔCT values shown in the bottom panels. Each data point is the mean ± SEM for all the mice examined at each time point.

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