American Society of Hematology

Figure 5.

$Figure 5. LXR activation in BPDCN cells interferes with IL-3–induced STAT5 and Akt phosphorylation, as well as NF-κB activation. (A) CAL-1 cells were treated with increasing concentrations (1 µM, 5 µM, or 10 µM) of LXR agonists (T09 or GW) for 24 hours, followed by 10 ng/mL of IL-3 for 30 minutes. Phospho-STAT5 (Y694), phospho-Akt (S473), as well as unphosphorylated corresponding protein expression was assessed by western blot. The expression of these proteins was compared with actin with the vehicle condition being considered as 1. Results of 1 representative experiment of 2. (B) BPDCN cells (LPDC #5) were treated with 1 µM LXR agonists for 24 hours. Phospho-STAT5 (Y694) and phospho-Akt (S473) were assessed by confocal microscopy after IL-3 stimulation (10 ng/mL, 30 minutes). Results of 1 representative sample (LPDC #5) of 3 BPDCN samples tested (LPDC #5, #6, #9). (C) CAL-1 and GEN2.2 cells were treated with increasing concentrations (12.5 µM, 25 µM, 50 µM, and 100 µM) of NF-κB inhibitor JSH23. Cell death was assessed by AnxV/7AAD staining, and analyzed by cytometry. Dot plots from 1 representative experiment of 5 and cumulative data from these 5 experiments expressed as percentage of dead AnxV+ cells are shown (left panel). Cumulative data from 3 experiments expressed as mean ± SEM are shown (right panel) (**P < .01, Mann-Whitney). (D) Primary BPDCN cells were treated with 1 µM of LXR agonists for 24 hours, followed by 1 µg/mL of R848 for 45 minutes. P65 phosphorylation (pS536) was assessed by confocal microscopy. Data from 1 representative primary BPDCN sample of 4 (LPDC #5, #6, #8, #9) are shown. (E) CAL-1 cells were treated with 1 µM T0901317 (T09) or GW3965 (GW) for 24 hours, followed by R848 (1 µg/mL) stimulation for 6 hours. Cytosolic and nuclear protein fractions were isolated as described in “Methods”. NF-κB1 (p105), p50, p65, and c-Rel proteins were analyzed by western blot and the expression of these proteins was compared with actin and lamin B expression (for cytosolic or nuclear expression, respectively) with the vehicle condition being considered as 1. Results from 1 representative experiment of 3.$

LXR activation in BPDCN cells interferes with IL-3–induced STAT5 and Akt phosphorylation, as well as NF-κB activation. (A) CAL-1 cells were treated with increasing concentrations (1 µM, 5 µM, or 10 µM) of LXR agonists (T09 or GW) for 24 hours, followed by 10 ng/mL of IL-3 for 30 minutes. Phospho-STAT5 (Y694), phospho-Akt (S473), as well as unphosphorylated corresponding protein expression was assessed by western blot. The expression of these proteins was compared with actin with the vehicle condition being considered as 1. Results of 1 representative experiment of 2. (B) BPDCN cells (LPDC #5) were treated with 1 µM LXR agonists for 24 hours. Phospho-STAT5 (Y694) and phospho-Akt (S473) were assessed by confocal microscopy after IL-3 stimulation (10 ng/mL, 30 minutes). Results of 1 representative sample (LPDC #5) of 3 BPDCN samples tested (LPDC #5, #6, #9). (C) CAL-1 and GEN2.2 cells were treated with increasing concentrations (12.5 µM, 25 µM, 50 µM, and 100 µM) of NF-κB inhibitor JSH23. Cell death was assessed by AnxV/7AAD staining, and analyzed by cytometry. Dot plots from 1 representative experiment of 5 and cumulative data from these 5 experiments expressed as percentage of dead AnxV⁺ cells are shown (left panel). Cumulative data from 3 experiments expressed as mean ± SEM are shown (right panel) (**P < .01, Mann-Whitney). (D) Primary BPDCN cells were treated with 1 µM of LXR agonists for 24 hours, followed by 1 µg/mL of R848 for 45 minutes. P65 phosphorylation (pS536) was assessed by confocal microscopy. Data from 1 representative primary BPDCN sample of 4 (LPDC #5, #6, #8, #9) are shown. (E) CAL-1 cells were treated with 1 µM T0901317 (T09) or GW3965 (GW) for 24 hours, followed by R848 (1 µg/mL) stimulation for 6 hours. Cytosolic and nuclear protein fractions were isolated as described in “Methods”. NF-κB1 (p105), p50, p65, and c-Rel proteins were analyzed by western blot and the expression of these proteins was compared with actin and lamin B expression (for cytosolic or nuclear expression, respectively) with the vehicle condition being considered as 1. Results from 1 representative experiment of 3.

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