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Figure 3. Scl/Tal1 regulates CD44 expression. (A) Volcano plot showing up- or down-regulated genes in K562 cells after knockdown of SCL/TAL1 relative to knockdown of lacZ as a control. The x-axis indicates the fold change and the y-axis indicates the −log10 P value. The orange and blue highlighted regions show 2-fold up- and down-re- gulated genes, respectively, with a P value ≤0.05. CD44 has been circled. (B, C) Relative expression of CD44 in K562 cells after infection with a SCL/TAL1 shRNA-express- ing or empty vector control (P=0.0001, t-test) (B) or an empty vector control- or a SCL/TAL1-overexpressing lentivirus (P<0.0001, t-test) (C). (D) Relative luminescence units (RLU) in a luciferase assay of K562 cells transfected with empty vector (control, black) or a plasmid expressing the CD44 regulatory element alone [P=0.039, analysis of variance (ANOVA); Tukey test], or the same CD44-regulatory element-transfected K562 cells transduced with a SCL/TAL1-overexpressing lentivirus (P=0.02, ANOVA; Tukey test) or transduced with two different shSCL/TAL1-expressing lentiviruses (P=0.049 and P=0.072, ANOVA; Tukey test, n=4). (E) Binding of SCL/TAL1 to the CD44 regulatory element in K562 cells treated with vehicle or imatinib as measured by a chromatin immunoprecipitation assay using an anti-SCL/TAL1 (white) or a control IgG (black) anti- body and four different CD44 primer pairs (P1-P4). (F) Kaplan-Meier-style survival curve for Balb/c recipients of BCR-ABL1-transduced bone marrow (BM) (solid line) or BCR-ABL1-transduced BM cotransduced with empty vector (dotted line) or cotransduced with an Scl/Tal1-overexpressing lentivirus (dashed line) (P=0.013, log-rank test, n=8). (G) Median fluorescence intensity (MFI) of CD44 on BCR-ABL1+ CD11b+ myeloid cells from recipients of BCR-ABL1-transduced BM (black) or BCR-ABL1-transduced BM cotransduced with empty vector (black) or cotransduced with a Scl/Tal1-overexpressing vector (white) as in (F) (P=0.031, t-test, n=6).
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