Figure 2.
SGK1 inhibition induces fetal hemoglobin in CD34+ cells from sickle cell donors and protects from sickling. Comp16y increases HbF and prevents sickling in differentiated CD34+ cells from sickle cell donors. Treatment with Comp16y increases HbF-positive erythroid cells differentiated from SCD donor blood peripheral blood mononuclear cell and treated cells resist sickling triggered by hypoxia (2% oxygen). (A) Treatment with Comp16y (5 μM) increases HbF-positive cells by day 14 of differentiation in representative flow cytometry plots from a single donor (left) and quantification of data from 3 independent sickle cell donors confirm a significant increase in HbF-positive cells (right) (3.5-fold increase). Two-tailed t test, ∗P < .05 (N=3). (B) Sickling under normoxia (20% oxygen) or hypoxia (2% oxygen) was assessed with fully differentiated CD34+ cells from SCD donor PBMC with and without SGK1 inhibition. Representative microscopic images of 21-day differentiated SCD donor CD34+ cells under hypoxia following differentiation in the presence of Comp16y (5 μM) or 0.1% dimethyl sulfoxide vehicle control (left) shows decreased sickling following SGK1 inhibition (2.1-fold decrease) (scale bar is 20 μm). Arrows indicate abnormal shaped cells. Similar results obtained for 3 independent donors. Sickle cell imaging flow cytometry assays were performed using the Amnis ImageStream and cell shape modification was quantitated using IDEAS software to quantify the percentage of sickling cells (middle) as described previously.21 Representative images by sickle cell imaging flow cytometry assays (right) of cell populations under normoxia gated as under normoxia (nonsickled) or hypoxia (2% oxygen) gated as sickled (60× magnification). Mean ± standard error of the mean (N = 3 independent HbSS SCD donors) and ∗P < .05 (two-tailed t test). (C) Flow cytometric analysis of CD235a+ cells on day 21 indicates normal differentiation of CD34+ cells treated with Comp16y (5 μM). Panels on left are representative flow cytometry plots from a single donor and quantification of data from 3 independent sickle cell donors show no significant decrease in CD235+ cells (middle). Two-tailed t test, ns = not significant (N = 3). Western blot analysis and quantification of selected erythroid differentiation markers (Band-3, LRF, ALAS2, GATA-1) on day 14 are all similar between Comp16y and vehicle treated SCD CD34+ cells when normalized to α-tubulin, confirming normal erythroid cell differentiation. A representative western blot (right top) and statistical analysis of all data (right bottom) are shown. Two-tailed t test, ns = not significant, N = 3. (D) Enucleation observed for fully differentiated SCD CD34+ cells on day 21 for both Comp16y and vehicle treatments shows a minor but significant effect of SGK1 inhibition on enucleation. This minor decrease in enucleation is consistent with normal erythroid differentiation. Two-tailed t test, ∗∗P < .01, N = 3.

SGK1 inhibition induces fetal hemoglobin in CD34+ cells from sickle cell donors and protects from sickling. Comp16y increases HbF and prevents sickling in differentiated CD34+ cells from sickle cell donors. Treatment with Comp16y increases HbF-positive erythroid cells differentiated from SCD donor blood peripheral blood mononuclear cell and treated cells resist sickling triggered by hypoxia (2% oxygen). (A) Treatment with Comp16y (5 μM) increases HbF-positive cells by day 14 of differentiation in representative flow cytometry plots from a single donor (left) and quantification of data from 3 independent sickle cell donors confirm a significant increase in HbF-positive cells (right) (3.5-fold increase). Two-tailed t test, ∗P < .05 (N=3). (B) Sickling under normoxia (20% oxygen) or hypoxia (2% oxygen) was assessed with fully differentiated CD34+ cells from SCD donor PBMC with and without SGK1 inhibition. Representative microscopic images of 21-day differentiated SCD donor CD34+ cells under hypoxia following differentiation in the presence of Comp16y (5 μM) or 0.1% dimethyl sulfoxide vehicle control (left) shows decreased sickling following SGK1 inhibition (2.1-fold decrease) (scale bar is 20 μm). Arrows indicate abnormal shaped cells. Similar results obtained for 3 independent donors. Sickle cell imaging flow cytometry assays were performed using the Amnis ImageStream and cell shape modification was quantitated using IDEAS software to quantify the percentage of sickling cells (middle) as described previously.21 Representative images by sickle cell imaging flow cytometry assays (right) of cell populations under normoxia gated as under normoxia (nonsickled) or hypoxia (2% oxygen) gated as sickled (60× magnification). Mean ± standard error of the mean (N = 3 independent HbSS SCD donors) and ∗P < .05 (two-tailed t test). (C) Flow cytometric analysis of CD235a+ cells on day 21 indicates normal differentiation of CD34+ cells treated with Comp16y (5 μM). Panels on left are representative flow cytometry plots from a single donor and quantification of data from 3 independent sickle cell donors show no significant decrease in CD235+ cells (middle). Two-tailed t test, ns = not significant (N = 3). Western blot analysis and quantification of selected erythroid differentiation markers (Band-3, LRF, ALAS2, GATA-1) on day 14 are all similar between Comp16y and vehicle treated SCD CD34+ cells when normalized to α-tubulin, confirming normal erythroid cell differentiation. A representative western blot (right top) and statistical analysis of all data (right bottom) are shown. Two-tailed t test, ns = not significant, N = 3. (D) Enucleation observed for fully differentiated SCD CD34+ cells on day 21 for both Comp16y and vehicle treatments shows a minor but significant effect of SGK1 inhibition on enucleation. This minor decrease in enucleation is consistent with normal erythroid differentiation. Two-tailed t test, ∗∗P < .01, N = 3.

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