American Society of Hematology

Figure 6

$Figure 6. BCL-xL upregulation via THPO/MPL signaling supports the growth and survival of CD41+ Evi1 leukemia cells. (A) Comparison of Bcl-xL (left), Bcl-2 (middle), and Mcl-1 (right) mRNA expression between CD41+ and CD41− BM cells of Evi1 leukemia mice. CD41+ and CD41− BM cells were sorted from 5 independent mice. Expression levels relative to normal c-kit+ BM cells are presented. Error bars indicate SD (*P < .05, Student t test). (B-D) Comparison of protein expression of BCL-xL (B), BCL-2 (C), and MCL-1 (D) between CD41+ and CD41− fractions by western blotting. Representative images and bar graphs showing quantified protein levels are presented. Expression levels were normalized to β-actin expression as the internal control and represented as relative values to those of CD41− cells. Quantification was performed by ImageJ software. Error bars indicate SD (n = 5; **P < .01, Student t test). (E-G) Cryopreserved BM- or SP-MNCs from Evi1 leukemia mice were thawed and serum-starved in α-MEM containing 1% BSA for 3 hours, and then stimulated with or without THPO in α-MEM containing 0.1% BSA for 7 hours. Cells were washed with phosphate-buffered saline and lysed for protein extraction. The expression levels of BCL-xL (E), BCL-2 (F), and MCL-1 (G) were determined by western blotting. Representative images and bar graphs showing quantified protein levels are presented. Quantification was performed as described above. Error bars indicate SD (n = 4; *P < .05, Student t test). (H-I) CD41+ or CD41− cells were treated with DMSO as a vehicle control or a BCL-xL inhibitor (WEHI-539; 0.2, 0.4, and 0.8 μM) on OP9 stromal cells in the presence of THPO for 7 days. (H) The number of viable cells was counted. Error bars indicate SD (n = 3). (I) The rate of apoptotic cells was determined by FACS. Error bars indicate SD (n = 3). Black and white bars represent the results from CD41+ and CD41− cells, respectively. There was no significant difference between DMSO- and WEHI-539-treated groups (Dunnett’s test). The concentration of THPO used in these experiments was 50 ng/mL.$

BCL-xL upregulation via THPO/MPL signaling supports the growth and survival of CD41⁺ Evi1 leukemia cells. (A) Comparison of Bcl-xL (left), Bcl-2 (middle), and Mcl-1 (right) mRNA expression between CD41⁺ and CD41⁻ BM cells of Evi1 leukemia mice. CD41⁺ and CD41⁻ BM cells were sorted from 5 independent mice. Expression levels relative to normal c-kit⁺ BM cells are presented. Error bars indicate SD (*P < .05, Student t test). (B-D) Comparison of protein expression of BCL-xL (B), BCL-2 (C), and MCL-1 (D) between CD41⁺ and CD41⁻ fractions by western blotting. Representative images and bar graphs showing quantified protein levels are presented. Expression levels were normalized to β-actin expression as the internal control and represented as relative values to those of CD41⁻ cells. Quantification was performed by ImageJ software. Error bars indicate SD (n = 5; **P < .01, Student t test). (E-G) Cryopreserved BM- or SP-MNCs from Evi1 leukemia mice were thawed and serum-starved in α-MEM containing 1% BSA for 3 hours, and then stimulated with or without THPO in α-MEM containing 0.1% BSA for 7 hours. Cells were washed with phosphate-buffered saline and lysed for protein extraction. The expression levels of BCL-xL (E), BCL-2 (F), and MCL-1 (G) were determined by western blotting. Representative images and bar graphs showing quantified protein levels are presented. Quantification was performed as described above. Error bars indicate SD (n = 4; *P < .05, Student t test). (H-I) CD41⁺ or CD41⁻ cells were treated with DMSO as a vehicle control or a BCL-xL inhibitor (WEHI-539; 0.2, 0.4, and 0.8 μM) on OP9 stromal cells in the presence of THPO for 7 days. (H) The number of viable cells was counted. Error bars indicate SD (n = 3). (I) The rate of apoptotic cells was determined by FACS. Error bars indicate SD (n = 3). Black and white bars represent the results from CD41⁺ and CD41⁻ cells, respectively. There was no significant difference between DMSO- and WEHI-539-treated groups (Dunnett’s test). The concentration of THPO used in these experiments was 50 ng/mL.

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