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. 2014 Dec 22;3(12):e135. doi: 10.1038/oncsis.2014.49

Figure 2.

Figure 2

HoxA9 and HoxA10 are antagonists for CDX4 promoter activity during myelopoiesis. (a) HoxA9 antagonizes the effect of HoxA10 on the CDX4 promoter. U937 cells were transfected with a reporter construct with 150 bp of CDX4 promoter and combinations of vectors to overexpress or knockdown HoxA9 or HoxA10. Reporter activity was determined with or without differentiation. Statistically significant differences in reporter activity are indicated by *, **, ***, #, ##, ### for with versus without overexpressed HoxA9; @, @@, @@@ or + for with versus without HoxA9 shRNA; ++, +++, $ or $$ for with versus without HoxA10 overexpression. (b) HoxA9 and HoxA10 protein are equivalently overexpressed in U937 transfectants. U937 cells were transfected with vectors to overexpress HoxA9, HoxA10, HoxA9+HoxA10 or control expression vector. Western blots were serially probed with antibodies to HoxA9, HoxA10 and Gapdh (as a loading control). (c) Tyrosine phosphorylation decreases the repression of the CDX4 promoter by HoxA9 and increases activation by HoxA10. U937 cells were transfected with a reporter construct with 150 bp of CDX4 promoter and vectors to overexpress tyrosine mutant HoxA9 (HD-Y-mutant HoxA9) or tyrosine mutant HoxA10 (HD-Y-mutant HoxA10) or vectors to overexpress wild-type or HD-Y-mutant HoxA9 or HoxA10 plus constitutively active Shp2 (E76K). Reporter activity was determined with or without differentiation. Statistically significant differences in reporter activity with wild-type versus tyrosine mutant protein are indicated by *, **, *** or #. Some differences that are not statistically significant (P>0.1) are indicated. (d) Constitutively active Jak2 increases repression of the CDX4 promoter by HoxA9 and decreases activation by HoxA10. U937 cells were transfected with a reporter construct with 150 bp of CDX4 promoter and a vector to express constitutively active Jak2 (V617F) with or without vectors to overexpress HoxA9, HoxA10 or both (or control vector). Statistically significant differences in reporter activity with HoxA9 or HoxA10 versus control vector are indicated by * or **. Statistically significant differences with versus without V617F-Jak2 are indicated by ***, # or ##. (e) Inhibition of Jak2 blocks repression of the CDX4 promoter by HoxA9 but increases activation by HoxA10 in differentiating myeloid cells, but inhibition of Src or Abl tyrosine kinase do not have a similar effect. U937 cells were transfected with a reporter construct with 150 bp of CDX4 promoter and vectors to overexpress HoxA9 or HoxA10 (or control vector). Cells were differentiated with retinoic acid/dimethyl formamide (RA/DMF) and some were treated with AZD 1480 (Jak2 inhibitor), Sarcatinib (Src inhibitor) or Imatinib (Ableson kinase inhibitor). Statistically significant differences in reporter activity with HoxA9 or HoxA10 overexpression versus control vector are indicated by * and **. Statistically significant differences with versus without Jak2-inhibition are indicated by *** or #.