Figure 1 Implication of PKD in... | American Society of Hematology

Figure 1

$Figure 1. Implication of PKD in erythropoiesis. (A) G1ER proerythroblasts underwent differentiation induction with erythropoietin (Epo), estradiol, and kinase inhibitors; percentage hemoglobinized cells was assessed by benzidine staining. Data are mean ± SEM for 3 independent experiments. (B) G1ER cells transduced with shRNA constructs targeting PKD3 were assessed for PKD3 expression, class IIa HDAC phosphorylation, and erythroid differentiation. sh 186 provided strong knockdown of PKD3 expression; sh 187 provided no knockdown; and sh 188 provided weak knockdown. Left: Immunoblot (IB) of transduced cells (8% gel). Right: Differentiation induction with estradiol and erythropoietin (0.5, 0.1, or 0.05 U/mL), showing mean ± SEM for 3 experiments. *P < .05 for sh 186 vs controls at corresponding Epo doses. (C) Human primary progenitors cultured 5 days in erythroid medium with 100% or 15% transferrin saturation. Kinase inhibitors were included at 2μM in medium with 100% transferrin saturation. Flow cytometry for the erythroid marker glycophorin A (GPA) and the megakaryocyte marker CD41, with gating on viable fractions by forward (FSC) and side scatter (SSC). See also supplemental Figure 1. (D) Class IIa HDAC phosphorylation in human progenitors cultured 5 days in erythroid medium with 100% or 15% transferrin saturation (TSAT) or with kinase inhibitors (0.5, 1.0, and 2.0μM, all with 100% transferrin saturation). IB of whole cell lysates (12% gel).$

Implication of PKD in erythropoiesis. (A) G1ER proerythroblasts underwent differentiation induction with erythropoietin (Epo), estradiol, and kinase inhibitors; percentage hemoglobinized cells was assessed by benzidine staining. Data are mean ± SEM for 3 independent experiments. (B) G1ER cells transduced with shRNA constructs targeting PKD3 were assessed for PKD3 expression, class IIa HDAC phosphorylation, and erythroid differentiation. sh 186 provided strong knockdown of PKD3 expression; sh 187 provided no knockdown; and sh 188 provided weak knockdown. Left: Immunoblot (IB) of transduced cells (8% gel). Right: Differentiation induction with estradiol and erythropoietin (0.5, 0.1, or 0.05 U/mL), showing mean ± SEM for 3 experiments. *P < .05 for sh 186 vs controls at corresponding Epo doses. (C) Human primary progenitors cultured 5 days in erythroid medium with 100% or 15% transferrin saturation. Kinase inhibitors were included at 2μM in medium with 100% transferrin saturation. Flow cytometry for the erythroid marker glycophorin A (GPA) and the megakaryocyte marker CD41, with gating on viable fractions by forward (FSC) and side scatter (SSC). See also supplemental Figure 1. (D) Class IIa HDAC phosphorylation in human progenitors cultured 5 days in erythroid medium with 100% or 15% transferrin saturation (TSAT) or with kinase inhibitors (0.5, 1.0, and 2.0μM, all with 100% transferrin saturation). IB of whole cell lysates (12% gel).

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