Figure 3
Figure 3. CYCLIN G2 protein levels are down-modulated by EPO, and ectopically expressed CYCLIN G2 attenuates UT7epo cell growth and S-phase transition. (A) EPO down-modulation of CYCLIN G2 protein expression. In primary wt-EPOR erythroblasts, EPO's effects on CYCLIN G2 expression levels were assayed by Western blotting, and first were observed to increase 2-fold or more within 6 hours of EPO withdrawal (top subpanels). In addition, EPO expression (in time-course and EPO concentration experiments) was observed to decrease CYCLIN G2 levels (bottom subpanels). Error bars represent standard deviations from the mean for duplicate scans of two ECL exposures (and are normalized for beta-tubulin). (B) Ectopic expression of CYCLIN G2 attenuates proliferation and G1- to S-phase transition in EPO-dependent UT7epo cells. UT7epo cells were transduced with a pBABEpuro-CYCLIN G2 retrovirus (or empty control vector) and were selected in puromycin. Derived lines (UT7epo CYCLIN G2 and UT7epo-vec) then were analyzed for EPO-dependent proliferative capacities and BrdU incorporation profiles. Ectopic expression of CYCLIN G2 first was observed to attenuate EPO-dependent UT7epo cell growth. Here, the cell lines were plated in the presence of EPO at 1 U/mL. Through time, viable cell numbers were monitored. Data shown are representative of 2 independent experiments. Subsequently, time-course analyses of EPO-dependent BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were performed. EPO was withdrawn from cultures for a 16-hour period prior to restimulation with EPO (1 U/mL) in the presence of BrdU. At the indicated time points, levels of BrdU incorporation were determined (top right subpanels). (C) Finally, EPO dose–dependent profiles of BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were investigated. Here, BrdU experiments were performed but with varying concentrations of EPO exposure. Panels first show raw data for incorporation levels. Bottom panels set gates for low versus high BrdU incorporation populations (gates 2 and 3, respectively), and further illustrate differences in BrdU incorporation profiles for gate 2 and 3 subpopulations for UT7epo CYCLIN G2 and UT7epo-vec cells.

CYCLIN G2 protein levels are down-modulated by EPO, and ectopically expressed CYCLIN G2 attenuates UT7epo cell growth and S-phase transition. (A) EPO down-modulation of CYCLIN G2 protein expression. In primary wt-EPOR erythroblasts, EPO's effects on CYCLIN G2 expression levels were assayed by Western blotting, and first were observed to increase 2-fold or more within 6 hours of EPO withdrawal (top subpanels). In addition, EPO expression (in time-course and EPO concentration experiments) was observed to decrease CYCLIN G2 levels (bottom subpanels). Error bars represent standard deviations from the mean for duplicate scans of two ECL exposures (and are normalized for beta-tubulin). (B) Ectopic expression of CYCLIN G2 attenuates proliferation and G1- to S-phase transition in EPO-dependent UT7epo cells. UT7epo cells were transduced with a pBABEpuro-CYCLIN G2 retrovirus (or empty control vector) and were selected in puromycin. Derived lines (UT7epo CYCLIN G2 and UT7epo-vec) then were analyzed for EPO-dependent proliferative capacities and BrdU incorporation profiles. Ectopic expression of CYCLIN G2 first was observed to attenuate EPO-dependent UT7epo cell growth. Here, the cell lines were plated in the presence of EPO at 1 U/mL. Through time, viable cell numbers were monitored. Data shown are representative of 2 independent experiments. Subsequently, time-course analyses of EPO-dependent BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were performed. EPO was withdrawn from cultures for a 16-hour period prior to restimulation with EPO (1 U/mL) in the presence of BrdU. At the indicated time points, levels of BrdU incorporation were determined (top right subpanels). (C) Finally, EPO dose–dependent profiles of BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were investigated. Here, BrdU experiments were performed but with varying concentrations of EPO exposure. Panels first show raw data for incorporation levels. Bottom panels set gates for low versus high BrdU incorporation populations (gates 2 and 3, respectively), and further illustrate differences in BrdU incorporation profiles for gate 2 and 3 subpopulations for UT7epo CYCLIN G2 and UT7epo-vec cells.

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