Figure 4.
Figure 4. Genome changes in AML cells following CDK2 depletion. (A-F) Microarray analysis of the gene expression changes in NB4 cells infected with lentivirus-shCDK2 (#1, #2, and #3) for 2 and 5 days. NB4 cells were subjected to GeneChip PrimerView Human Gene Expression Array for gene expression analyzes. (A) The mRNA changes of CDK2 and differentiation marker genes (C/EBPB, PU.1, STAT1, CD11b, CD18, IL1B, and CD14) generated from microarray analysis. (B) Validation of the mRNA changes of C/EBPB and PU.1 by real-time PCR. (C) Schematic representation of comparing gene expression profiles in NB4 cells. (Top) Overlapped smaller circles reflect the shared genes induced by different lentivirus-shCDK2s on day 2 (left; blue) and day 5 (right; purple), respectively. Top left, 35 genes changed on day 2 (up: 18 vs down: 17). Top right, 499 genes changed on day 5 (up: 473 vs down: 26). (Bottom) Overlapped smaller oval reflect the shared 16 genes induced on both day 2 and day 5. Red numbers in circles reflect upregulation, while green numbers reflect downregulation. (D) Heat map display of hierarchical clustering of overlapped genes sorted from top panel, (C). A total of 518 genes whose expression changed more than twofold on day 2 or day 5 were clustered. (E) (C) Heat map display of hierarchical clustering of overlapped 16 genes sorted from bottom panel. A total of 16 genes whose expression changed more than twofold both on days 2 and 5 were clustered. (F) Validation of 16 upregulated genes in (E) by real-time PCR. (G) The protein expression levels of MAFB and CDK2 in NB4 cells infected with lentivirus-shCDK2s for 2 or 5 days. (H-J) The effect of MAFB depletion on shCDK2-induced differentiation in NB4 cells. NB4 cells were infected with lentivirus-shMAFB (#3 and #4) for 1 day, followed by infection with lentivirus-shCDK2s for another 2 or 5 days. (H) Silencing efficiency of different shMAFBs. MAFB protein levels were evaluated by western blotting. (I) Percentage of CD11b expression. (J) NBT-reducing activity. (I-J) ++, P < .01; +++, P < .001; shMAFB vs scramble in the shCDK2 #1 group. #P < .05; ##P < .05; ###P < .001; shMAFB vs scramble in the shCDK2 #2 group. *P < .05; **P < .05; ***P < .001; shMAFB vs scramble in the shCDK2 #3 group. Data are shown as mean ± SD, n = 3. Other data are representative of at least 3 individual experiments; 1 representative image is shown.

Genome changes in AML cells following CDK2 depletion. (A-F) Microarray analysis of the gene expression changes in NB4 cells infected with lentivirus-shCDK2 (#1, #2, and #3) for 2 and 5 days. NB4 cells were subjected to GeneChip PrimerView Human Gene Expression Array for gene expression analyzes. (A) The mRNA changes of CDK2 and differentiation marker genes (C/EBPB, PU.1, STAT1, CD11b, CD18, IL1B, and CD14) generated from microarray analysis. (B) Validation of the mRNA changes of C/EBPB and PU.1 by real-time PCR. (C) Schematic representation of comparing gene expression profiles in NB4 cells. (Top) Overlapped smaller circles reflect the shared genes induced by different lentivirus-shCDK2s on day 2 (left; blue) and day 5 (right; purple), respectively. Top left, 35 genes changed on day 2 (up: 18 vs down: 17). Top right, 499 genes changed on day 5 (up: 473 vs down: 26). (Bottom) Overlapped smaller oval reflect the shared 16 genes induced on both day 2 and day 5. Red numbers in circles reflect upregulation, while green numbers reflect downregulation. (D) Heat map display of hierarchical clustering of overlapped genes sorted from top panel, (C). A total of 518 genes whose expression changed more than twofold on day 2 or day 5 were clustered. (E) (C) Heat map display of hierarchical clustering of overlapped 16 genes sorted from bottom panel. A total of 16 genes whose expression changed more than twofold both on days 2 and 5 were clustered. (F) Validation of 16 upregulated genes in (E) by real-time PCR. (G) The protein expression levels of MAFB and CDK2 in NB4 cells infected with lentivirus-shCDK2s for 2 or 5 days. (H-J) The effect of MAFB depletion on shCDK2-induced differentiation in NB4 cells. NB4 cells were infected with lentivirus-shMAFB (#3 and #4) for 1 day, followed by infection with lentivirus-shCDK2s for another 2 or 5 days. (H) Silencing efficiency of different shMAFBs. MAFB protein levels were evaluated by western blotting. (I) Percentage of CD11b expression. (J) NBT-reducing activity. (I-J) ++, P < .01; +++, P < .001; shMAFB vs scramble in the shCDK2 #1 group. #P < .05; ##P < .05; ###P < .001; shMAFB vs scramble in the shCDK2 #2 group. *P < .05; **P < .05; ***P < .001; shMAFB vs scramble in the shCDK2 #3 group. Data are shown as mean ± SD, n = 3. Other data are representative of at least 3 individual experiments; 1 representative image is shown.

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