Figure 3
Figure 3. shRNA knockdown of Id1 during in vitro erythroid differentiation. (A) Id1 transcript levels after 24 hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with shRNA constructs targeting the murine Id1 gene or firefly luciferase (to control for nonspecific cellular effects associated with the processing of an shRNA hairpin). (B) Expansion of lineage-negative E14.5 fetal liver cells during in vitro erythroid differentiation. Data (mean and standard deviation) for 1 of 3 independent experiments, each performed in triplicate and showing a similar result, are shown. (C) Relative increase in the number of lineage-negative E14.5 fetal liver cells transfected with knockdown constructs targeting Id1 or firefly luciferase during 48 hours of in vitro culture. The fold increase in the number of cells transfected with Id1 knockdown constructs after 48 hours of in vitro culture was normalized against that for cells transfected with the construct targeting firefly luciferase. Id1 knockdown is associated with a highly statistically significant 40% to 50% decrease in cell expansion during 48 hours of in vitro culture. Shown is the mean and SEM for 3 independent experiments, each performed in triplicate (**P < .01; ***P < .001; see “Results” for details). (D) Representative FACS plots for ter119 and CD71 expression after 24 and 48 hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with knockdown constructs targeting Id1 or luciferase. The lefthand panels (0-hours time point) illustrate an identical pool of purified lineage-negative fetal liver cells before retroviral transduction with luciferase or Id1 knockdown constructs and are replicated to facilitate comparison with the panels to their right. Bar charts indicating the proportion of cells at each of the 5 stages of erythroid differentiation (R1-R5) after 24 (E) and 48 (F) hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with knockdown constructs targeting Id1 or luciferase. Data (mean and SD) for 1 of 3 independent experiments, each performed in triplicate and showing a similar result, are shown.

shRNA knockdown of Id1 during in vitro erythroid differentiation. (A) Id1 transcript levels after 24 hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with shRNA constructs targeting the murine Id1 gene or firefly luciferase (to control for nonspecific cellular effects associated with the processing of an shRNA hairpin). (B) Expansion of lineage-negative E14.5 fetal liver cells during in vitro erythroid differentiation. Data (mean and standard deviation) for 1 of 3 independent experiments, each performed in triplicate and showing a similar result, are shown. (C) Relative increase in the number of lineage-negative E14.5 fetal liver cells transfected with knockdown constructs targeting Id1 or firefly luciferase during 48 hours of in vitro culture. The fold increase in the number of cells transfected with Id1 knockdown constructs after 48 hours of in vitro culture was normalized against that for cells transfected with the construct targeting firefly luciferase. Id1 knockdown is associated with a highly statistically significant 40% to 50% decrease in cell expansion during 48 hours of in vitro culture. Shown is the mean and SEM for 3 independent experiments, each performed in triplicate (**P < .01; ***P < .001; see “Results” for details). (D) Representative FACS plots for ter119 and CD71 expression after 24 and 48 hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with knockdown constructs targeting Id1 or luciferase. The lefthand panels (0-hours time point) illustrate an identical pool of purified lineage-negative fetal liver cells before retroviral transduction with luciferase or Id1 knockdown constructs and are replicated to facilitate comparison with the panels to their right. Bar charts indicating the proportion of cells at each of the 5 stages of erythroid differentiation (R1-R5) after 24 (E) and 48 (F) hours of in vitro culture of lineage-negative E14.5 fetal liver cells retrovirally transfected with knockdown constructs targeting Id1 or luciferase. Data (mean and SD) for 1 of 3 independent experiments, each performed in triplicate and showing a similar result, are shown.

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