Figure 5.
Expression of embryonic stem cell markers in AML cells can be enhanced by CXCL12, depends on CD44 expression, and is associated to apoptosis resistance. (A) OCI-AML3 cells express core ESC-TFs. (Ai) Modified version of the PL-SIN-EOS-S(4+)-EGFP pluripotency reporter23 (Addgene MTA [Order 35560]), expressing the unstable version of EGFP (d2EGFP: half-life, 2 hours), followed by the constitutive promoter SV40 driving the puromycin resistance gene, which allows the selection of cells carrying the reporter independently from their Oct4/Sox2 expression levels. (Aii) OCI-AML3 cells transduced with the vector described in panel Ai OCI-AML3-EOS-S(4+)d2EGFP were analyzed by FACS for the expression of d2EGFP (parental OCI-AML3 cells were used as the negative control for the gating strategy), and (Aiii) sorted for cells expressing the reporter (d2EGFP+) and cells not expressing it (d2EGFP–). (B) CXCL12 enhanced ESC-TF expression requires CD44. (Bi) OCI-AML3-EOS-S(4+)d2EGFP cells transfected with CD44 siRNA or control siRNA were treated with CXCL12 (200 ng/mL) for 1 hour or left untreated. Percentages of d2EGFP+ cells were measured by FACS (Wilcoxon rank-sum test for 3 independent experiments). (Bii) Parental OCI-AML3 cells were treated as in panel Bi, and mRNA levels of the core ESC-TFs were measured by qPCR (1-way ANOVA of 3 independent experiments). (Biii) Primary cells from 3 AML patient samples were treated with CXCL12 (200 ng/mL), and mRNA levels of the core ESC-TFs were measured by qPCR. (Ci) mRNA levels of CD44, CXCR4, and BCL-2 family members were determined by qPCR and expressed as fold change between cells sorted for d2EGFP expression as in panel Aiii. (Cii) OCI-AML3-EOS-S(4+)d2EGFP and Molm13-EOS-S(4+)d2EGFP cells were treated with venetoclax 3 μM for 16 hours, and the percentage of d2EGFP+ was measured by FACS. (D) Molm13-VR-EOS-S(4+)d2EGFP and OCI-AML3-EOS-S(4+)d2EGFP cells were sorted for the ∼20% of cells expressing the highest and the ∼20% expressing the lowest d2EGFP fluorescence intensity and were treated with venetoclax or DMSO. Apoptotic (annexin V+/PI+) cells were determined by flow cytometry; annexin V and PI staining was used. Three independent experiments were quantified with a Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. GFP, green fluorescent protein; SSC-H, side scatter height.

Expression of embryonic stem cell markers in AML cells can be enhanced by CXCL12, depends on CD44 expression, and is associated to apoptosis resistance. (A) OCI-AML3 cells express core ESC-TFs. (Ai) Modified version of the PL-SIN-EOS-S(4+)-EGFP pluripotency reporter23 (Addgene MTA [Order 35560]), expressing the unstable version of EGFP (d2EGFP: half-life, 2 hours), followed by the constitutive promoter SV40 driving the puromycin resistance gene, which allows the selection of cells carrying the reporter independently from their Oct4/Sox2 expression levels. (Aii) OCI-AML3 cells transduced with the vector described in panel Ai OCI-AML3-EOS-S(4+)d2EGFP were analyzed by FACS for the expression of d2EGFP (parental OCI-AML3 cells were used as the negative control for the gating strategy), and (Aiii) sorted for cells expressing the reporter (d2EGFP+) and cells not expressing it (d2EGFP–). (B) CXCL12 enhanced ESC-TF expression requires CD44. (Bi) OCI-AML3-EOS-S(4+)d2EGFP cells transfected with CD44 siRNA or control siRNA were treated with CXCL12 (200 ng/mL) for 1 hour or left untreated. Percentages of d2EGFP+ cells were measured by FACS (Wilcoxon rank-sum test for 3 independent experiments). (Bii) Parental OCI-AML3 cells were treated as in panel Bi, and mRNA levels of the core ESC-TFs were measured by qPCR (1-way ANOVA of 3 independent experiments). (Biii) Primary cells from 3 AML patient samples were treated with CXCL12 (200 ng/mL), and mRNA levels of the core ESC-TFs were measured by qPCR. (Ci) mRNA levels of CD44, CXCR4, and BCL-2 family members were determined by qPCR and expressed as fold change between cells sorted for d2EGFP expression as in panel Aiii. (Cii) OCI-AML3-EOS-S(4+)d2EGFP and Molm13-EOS-S(4+)d2EGFP cells were treated with venetoclax 3 μM for 16 hours, and the percentage of d2EGFP+ was measured by FACS. (D) Molm13-VR-EOS-S(4+)d2EGFP and OCI-AML3-EOS-S(4+)d2EGFP cells were sorted for the ∼20% of cells expressing the highest and the ∼20% expressing the lowest d2EGFP fluorescence intensity and were treated with venetoclax or DMSO. Apoptotic (annexin V+/PI+) cells were determined by flow cytometry; annexin V and PI staining was used. Three independent experiments were quantified with a Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. GFP, green fluorescent protein; SSC-H, side scatter height.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal