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CD99 overexpression in AML
S7A). Similarly, quantitative polymerase chain reaction HD PBMC (n=3) and CD34+ cells. Transcript
(qPCR) analysis showed that CD99 transcripts were up- regulated in AML blast samples (n=9) and AML cell lines (KG-1a, U937, THP-1, and MOLM-13) compared with
ENST00000482405.7 is higher in CD34+ cells than in AML cell lines (Online Supplementary Figure S7B; for qPCR primers see Online Supplementary Table S5).
AB
CD
E
F
G
I
HI
Figure 3. Effect of CD99 isoform expression on acute myeloid leukemia (AML) cell proliferation. (A) Relative CD99 expression in cord blood cells (n=2) and AML cell lines (n=9) measured by flow cytometry. (B) Western blot analysis of CD99 isoform expression in CD34+ cells, healthy donor peripheral blood mononuclear cells (PBMC) and AML cell lines. (C) Viability of CD99-shRNA knockdown in THP-1, MOLM-13, U937 and AML-4 cells measured 96 hours (h) after transfection using try- pan-blue and alamar-blue. (D) Quantative polymerase chain reaction (qPCR) analysis using CD99 TaqMan assay to confirm CD99 knockdown in THP-1, MOLM-13, U937 and AML-4 cells transfected with CD99-shRNA plasmid (or EV) for 96 h. CD99 expression was normalized to EV cells. (E) CD99 expression in EV, CD99-L OE and CD99-S OE cells measured by flow cytometry in THP-1, U937 and MOLM-13 cells.(F) Proliferation assay of EV, CD99-L and CD99-S measured at 24,48 and 72 h by trypan-blue (n=4) in THP-1, U937, and MOLM-13 cells. Data reported as number of live cells. (G) Viability of AML blasts over-expressing CD99-L or EV measured 96 h after lentiviral transduction using trypan-blue (n=7). Data reported as total number of live cells/mL. (H) Viability of AML blasts over-expressing CD99 L, CD99- S or EV measured 96 h after lentiviral transduction using trypan-blue (n=3). Data reported as the number of live cells/mL. (I) Long-term proliferation assay of EV, CD99-L and CD99-S cells measured at days 2, 3, 4, 6, 7 and 9 by trypan-blue assay. (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05).
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