American Society of Hematology

Figure 4.

$Figure 4. LXR activation in BPDCN cells induces apoptotic cell death. CAL-1, GEN2.2, BES1, and blood samples from 5 patients diagnosed with BPDCN (LPDC #1, #2, #5, #10, #11) were treated with increasing concentrations (10-50 µM) of LXR agonists T0901317 (T09) or GW3965 (GW) for 24 hours. Cell viability was assessed by AnxV and 7AAD staining and cytometry. (A) Left panel, Dot plots illustrate data obtained with cells treated with 50 µM LXR agonists, except for CAL-1 cells that were treated with 10 µM and 50 µM. Results from 1 representative experiment of 6 for CAL-1, 1 of 4 for GEN2.2 cells, and 1 representative sample (LPDC#11) of 5 for primary BPDCN samples tested. Right panel, The percentage of AnxV+ dead cells (mean ± SEM from 6 or 4 independent experiments for CAL-1 and GEN2.2, respectively), after LXR agonist treatment (*P < .05, **P < .01, ***P < .001, ****P < .0001, Mann-Whitney). (B) One freshly isolated blood sample obtained from a patient diagnosed with BPDCN was treated with 50 µM T0901317 (T09) or GW3965 (GW) for 24 hours. Cytometry dot plots from 1 representative sample of 5 different BPDCN represent the percentage of BPDCN cells (identified by CD123/BDCA4 staining) in the viable cell fraction of blood sample (ie, the AnxV−/7AAD− fraction) (*P < .05, Mann-Whitney). (C) CAL-1 cells were treated with increasing concentrations of LXR agonists, T0901317 (T09) or GW3965 (GW), for 6 hours. Analysis of full-length inactive (inact.) and cleaved active (act.) forms of caspase-3 and caspase-9 was performed by western blot. Expression of these proteins was compared with actin expression with the vehicle condition being considered as 1. Results from 1 representative experiment of 3. (D) BPDCN cells (GEN2.2 and 2 primary BPDCN samples, LPDC #1 and #5) were treated with 50 µM T0901317 (T09) or GW3965 (GW) for 6 hours. Caspase-9 activation on 7AAD− cell fraction (excluding late apoptotic and necrotic cells) was assessed by cytometry. One dot plot represents 1 representative experiment of 2 for GEN2.2 cells, and the other 2 dot plots represent the experiments performed on 2 BPDCN samples (LPDC #1 and #5). (E) CAL-1 and GEN2.2 cells were treated with increasing concentrations of LXR agonists (10 µM, 30 µM, and 50 µM) for 6 hours. BCL2, BAX, and BAK1 gene expression was assessed by qRT-PCR. Levels of mRNA were normalized to those of GAPDH for each sample and then expressed as fold change related to the average value for vehicle-treated cells. Cumulative data from 4 independent experiments expressed as mean ± SEM (*P < .05, **P < .01, Mann-Whitney) are shown. FSC, forward scatter; SSC, side scatter.$

LXR activation in BPDCN cells induces apoptotic cell death. CAL-1, GEN2.2, BES1, and blood samples from 5 patients diagnosed with BPDCN (LPDC #1, #2, #5, #10, #11) were treated with increasing concentrations (10-50 µM) of LXR agonists T0901317 (T09) or GW3965 (GW) for 24 hours. Cell viability was assessed by AnxV and 7AAD staining and cytometry. (A) Left panel, Dot plots illustrate data obtained with cells treated with 50 µM LXR agonists, except for CAL-1 cells that were treated with 10 µM and 50 µM. Results from 1 representative experiment of 6 for CAL-1, 1 of 4 for GEN2.2 cells, and 1 representative sample (LPDC#11) of 5 for primary BPDCN samples tested. Right panel, The percentage of AnxV⁺ dead cells (mean ± SEM from 6 or 4 independent experiments for CAL-1 and GEN2.2, respectively), after LXR agonist treatment (*P < .05, **P < .01, ***P < .001, ****P < .0001, Mann-Whitney). (B) One freshly isolated blood sample obtained from a patient diagnosed with BPDCN was treated with 50 µM T0901317 (T09) or GW3965 (GW) for 24 hours. Cytometry dot plots from 1 representative sample of 5 different BPDCN represent the percentage of BPDCN cells (identified by CD123/BDCA4 staining) in the viable cell fraction of blood sample (ie, the AnxV⁻/7AAD⁻ fraction) (*P < .05, Mann-Whitney). (C) CAL-1 cells were treated with increasing concentrations of LXR agonists, T0901317 (T09) or GW3965 (GW), for 6 hours. Analysis of full-length inactive (inact.) and cleaved active (act.) forms of caspase-3 and caspase-9 was performed by western blot. Expression of these proteins was compared with actin expression with the vehicle condition being considered as 1. Results from 1 representative experiment of 3. (D) BPDCN cells (GEN2.2 and 2 primary BPDCN samples, LPDC #1 and #5) were treated with 50 µM T0901317 (T09) or GW3965 (GW) for 6 hours. Caspase-9 activation on 7AAD⁻ cell fraction (excluding late apoptotic and necrotic cells) was assessed by cytometry. One dot plot represents 1 representative experiment of 2 for GEN2.2 cells, and the other 2 dot plots represent the experiments performed on 2 BPDCN samples (LPDC #1 and #5). (E) CAL-1 and GEN2.2 cells were treated with increasing concentrations of LXR agonists (10 µM, 30 µM, and 50 µM) for 6 hours. BCL2, BAX, and BAK1 gene expression was assessed by qRT-PCR. Levels of mRNA were normalized to those of GAPDH for each sample and then expressed as fold change related to the average value for vehicle-treated cells. Cumulative data from 4 independent experiments expressed as mean ± SEM (*P < .05, **P < .01, Mann-Whitney) are shown. FSC, forward scatter; SSC, side scatter.

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