Figure 2
Figure 2. caSTAT5a inhibits the transcriptional activity of the LEF-1 promoter. (A) A LEF-1 reporter construct containing a 4000-bp upstream region of the LEF-1 gene harboring 3 binding sites identified in chromatin immunoprecipitation assays (top inset) was generated. The effects of exogenously expressed caSTAT5a, WT STAT5a, or STAT5a Y-F MUT on LEF-1 autoregulation were then tested in HEK293 cells co-transfected with the LEF-1 reporter construct. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (B) LEF-1 mRNA expression in HEK293 cells transfected as described in panel A. mRNA was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (C) The effects of caSTAT5a, WT STAT5a, or STAT5a Y-F MUT on the LEF-1–mediated activation of a TOP promoter construct containing 4 LEF-1/TCFs binding sites were analyzed in HEK 293 cells co-transfected with the TOP construct and the corresponding expression plasmids by measuring the luciferase activity of the TOP promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (D,E) The effects of caSTAT5a on the LEF-1–mediated activation of C/EBPα and cyclin D1 promoters were analyzed in HEK 293 cells co-transfected with cyclin D1 (D) or C/EBPα (E) reporter gene constructs and the corresponding expression plasmids. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (F) Three STAT5 binding sites were deleted in the LEF-1 gene promoter (top inset). caSTAT5a-dependent inhibition of LEF-1 promoter autoregulation was determined in HEK293 cells co-transfected with the promoter construct containing mutated STAT5 binding sites and the corresponding expression plasmids by assessing the luciferase activity of the mutated LEF-1 promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (**P < .01). (G) Five LEF-1 binding sites were deleted in the LEF-1 promoter (top inset) caSTAT5a-dependent inhibition of LEF-1 promoter autoregulation was determined in HEK293 cells co-transfected with the promoter construct containing mutated LEF-1 binding sites and the corresponding expression plasmids by assessing the luciferase activity of the mutated LEF-1 promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate.

caSTAT5a inhibits the transcriptional activity of the LEF-1 promoter. (A) A LEF-1 reporter construct containing a 4000-bp upstream region of the LEF-1 gene harboring 3 binding sites identified in chromatin immunoprecipitation assays (top inset) was generated. The effects of exogenously expressed caSTAT5a, WT STAT5a, or STAT5a Y-F MUT on LEF-1 autoregulation were then tested in HEK293 cells co-transfected with the LEF-1 reporter construct. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (B) LEF-1 mRNA expression in HEK293 cells transfected as described in panel A. mRNA was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (C) The effects of caSTAT5a, WT STAT5a, or STAT5a Y-F MUT on the LEF-1–mediated activation of a TOP promoter construct containing 4 LEF-1/TCFs binding sites were analyzed in HEK 293 cells co-transfected with the TOP construct and the corresponding expression plasmids by measuring the luciferase activity of the TOP promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (D,E) The effects of caSTAT5a on the LEF-1–mediated activation of C/EBPα and cyclin D1 promoters were analyzed in HEK 293 cells co-transfected with cyclin D1 (D) or C/EBPα (E) reporter gene constructs and the corresponding expression plasmids. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (F) Three STAT5 binding sites were deleted in the LEF-1 gene promoter (top inset). caSTAT5a-dependent inhibition of LEF-1 promoter autoregulation was determined in HEK293 cells co-transfected with the promoter construct containing mutated STAT5 binding sites and the corresponding expression plasmids by assessing the luciferase activity of the mutated LEF-1 promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (**P < .01). (G) Five LEF-1 binding sites were deleted in the LEF-1 promoter (top inset) caSTAT5a-dependent inhibition of LEF-1 promoter autoregulation was determined in HEK293 cells co-transfected with the promoter construct containing mutated LEF-1 binding sites and the corresponding expression plasmids by assessing the luciferase activity of the mutated LEF-1 promoter. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate.

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