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. 2011 Apr 6;286(21):19047–19064. doi: 10.1074/jbc.M110.213983

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 9. — Cdx4 influenced expression of HoxA10 target genes, and HoxA10 influenced expression of Cdx4 target genes in myeloid cells. A, HoxA10 overexpression influenced Cdx4 target gene expression. U937 cells were stably co-transfected with a vector to overexpress HoxA10 or Cdx4 (or empty expression vector) and a vector to express HoxA10- or Cdx4-specific shRNAs (or scrambled control shRNAs). Expression of HoxB3, HoxB4, and HoxA9 mRNA was determined by real-time PCR. *, ##, and &&&, statistically significant differences in expression of HoxB3, HoxB4, and HoxA9, respectively, with HoxA10 overexpression or knockdown in comparison with control (p < 0.00001, n = 6). **, ###, and +, statistically significant differences in HoxB3, HoxB4, and HoxA9 expression, respectively, in cells with Cdx4 overexpression or knockdown versus control cells (p < 0.0001, n = 6). ***, &, and ++, statistically significant increase in HoxB3, HoxB4, and HoxA9 mRNA, respectively, in cells with shHoxA10 upon overexpression of Cdx4 (p < 0.0001, n = 6). #, &&, and +++, statistically significant increase in HoxB3, HoxB4, and HoxA9, respectively, in cells with shCdx4 upon overexpression of HoxA10 (p < 0.0001, n = 6). B, Cdx4 overexpression influences HoxA10 target gene expression. The U937 stable transfectants described above were also analyzed for expression of gp91^PHOX, β3 integrin, Mkp2, or Tgfβ2 by real-time PCR. * and **, statistically significant increase in gp91^PHOX mRNA expression with HoxA10 knockdown and decrease with Cdx4 overexpression, respectively (p ≤ 0.03, n = 3). ***, increased gp91^PHOX mRNA expression in Cdx4-overexpressing cells with co-expression of HoxA10-specific shRNA (p < 0.001, n = 6). # and ##, statistically significant decrease in β3 integrin expression with HoxA10 knockdown and increase with Cdx4 overexpression, respectively (p ≤ 0.02, n = 3). ###, decreased β3 integrin expression in Cdx4-overexpressing cells with co-expression of HoxA10-specific shRNA (p < 0.001, n = 3). & and &&, statistically significant decrease in Mkp2 expression with HoxA10 knockdown and increase with Cdx4 overexpression, respectively (p ≤ 0.004, n = 3). &&&, decreased Mkp2 expression in Cdx4-overexpressing cells with co-expression of HoxA10-specific shRNA (p < 0.001, n = 4). + and ++, statistically significant decrease in Tgfβ2 expression with HoxA10 knockdown and increase with Cdx4 overexpression, respectively (p ≤ 0.03, n = 4). +++, decreased Tgfβ2 expression in Cdx4-overexpressing cells with co-expression of HoxA10-specific shRNA (p = 0.004, n = 4). C, Cdx4 knockdown decreased cytokine hypersensitivity of HoxA10-overexpressing cells. U937 cells were stably co-transfected with vectors to overexpress HoxA10-specific (or empty vector control) and Cdx4-specific shRNAs (or scrambled control shRNAs). Cells were deprived of cytokines, and proliferation was determined by [³H]thymidine incorporation after stimulation with a dose titration of fetal calf serum. *, statistically significant differences in [³H]thymidine incorporation in HoxA10-overexpressing transfectants with versus without Cdx4 knockdown (p < 0.01, n = 6). **, statistically significant difference in proliferation in HoxA10 overexpressing transfectants with knockdown of Cdx4 versus control transfectants (p < 0.01, n = 6). Error bars, S.E.