Figure 1.
Figure 1. NCAM1 is heterogeneously expressed in all AML subsets. (A) Quantitative mRNA expression of NCAM1 was assessed in 11 AML cell lines (blue) and 16 patient samples (red). Cycle threshold (CT) values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and log-fold expression is shown relative to the sample with lowest NCAM1 expression for each group. Cutoff values were arbitrarily chosen as <1 × 102 for NCAM−, 1 × 102-1 × 104 for NCAMlow, and >1 × 104 for NCAMhigh samples. (B) Cell surface expression of NCAM1 relative to isotypic immunoglobulin G (IgG) control in indicated AML cell lines (left panel) and patient samples (right panel) was analyzed by flow cytometry. Dotted lines separate NCAM1− from NCAM1+ samples. Error bars represent mean of 3 measurements ± SD of mean. (C) Correlation of NCAM1 mRNA and cell surface expression (log10 transformed values; Pearson correlation, r). (D-E) Analysis of NCAM1 expression across different cytogenetic (D) and molecular (E) groups derived from the indicated data sets. Analysis of variance (ANOVA) was significant at P < .01 in all comparisons. Shown P values represent significant differences between the corresponding group and mean values of all other groups. (F) Flow cytometry analysis of NCAM1 cell surface expression was performed 3 days after Dox-mediated induction of shRNA (shAF9_3′, shAF9_5′, shMLL-AF9 or Scr) as indicated. Shown is the mean fluorescence intensity (MFI) relative to Scr_shRNA controls and was compared by the 2-tailed Student t test. Error bars represent mean of 3 measurements ± SD of mean. (G) qPCR after DNAseI treatment at regions flanking indicated motifs near the NCAM1 transcription start site (TSS). The y-axis shows log2-fold changes (FC) between the Ct values at baseline (1 U) and increasing DNAseI concentrations (2 U and 4 U). Shown are results of 2 independent experimental replicates for each cell line. (H) Flow cytometry analysis of NCAM1 cell surface expression was performed 3 days after Dox-mediated induction of shRNA expression directed against STAT1, MEIS1, MEF2c, or Scr as indicated. Shown is the MFI relative to Scr_shRNA controls. Differences were compared using the 2-tailed Student t test. Error bars represent mean of 3 to 6 measurements ± SD of mean.

NCAM1 is heterogeneously expressed in all AML subsets. (A) Quantitative mRNA expression of NCAM1 was assessed in 11 AML cell lines (blue) and 16 patient samples (red). Cycle threshold (CT) values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and log-fold expression is shown relative to the sample with lowest NCAM1 expression for each group. Cutoff values were arbitrarily chosen as <1 × 102 for NCAM−, 1 × 102-1 × 104 for NCAMlow, and >1 × 104 for NCAMhigh samples. (B) Cell surface expression of NCAM1 relative to isotypic immunoglobulin G (IgG) control in indicated AML cell lines (left panel) and patient samples (right panel) was analyzed by flow cytometry. Dotted lines separate NCAM1− from NCAM1+ samples. Error bars represent mean of 3 measurements ± SD of mean. (C) Correlation of NCAM1 mRNA and cell surface expression (log10 transformed values; Pearson correlation, r). (D-E) Analysis of NCAM1 expression across different cytogenetic (D) and molecular (E) groups derived from the indicated data sets. Analysis of variance (ANOVA) was significant at P < .01 in all comparisons. Shown P values represent significant differences between the corresponding group and mean values of all other groups. (F) Flow cytometry analysis of NCAM1 cell surface expression was performed 3 days after Dox-mediated induction of shRNA (shAF9_3′, shAF9_5′, shMLL-AF9 or Scr) as indicated. Shown is the mean fluorescence intensity (MFI) relative to Scr_shRNA controls and was compared by the 2-tailed Student t test. Error bars represent mean of 3 measurements ± SD of mean. (G) qPCR after DNAseI treatment at regions flanking indicated motifs near the NCAM1 transcription start site (TSS). The y-axis shows log2-fold changes (FC) between the Ct values at baseline (1 U) and increasing DNAseI concentrations (2 U and 4 U). Shown are results of 2 independent experimental replicates for each cell line. (H) Flow cytometry analysis of NCAM1 cell surface expression was performed 3 days after Dox-mediated induction of shRNA expression directed against STAT1, MEIS1, MEF2c, or Scr as indicated. Shown is the MFI relative to Scr_shRNA controls. Differences were compared using the 2-tailed Student t test. Error bars represent mean of 3 to 6 measurements ± SD of mean.

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