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. 2020 Oct 26;8:587778. doi: 10.3389/fcell.2020.587778

FIGURE 7.

FIGURE 7

LMX1A-mediated activation of the murine Pitx3 promoter is suppressed in the presence of high LEF1/b-catenin levels. (A) RT-PCR analyses of total RNA isolated from untransfected HEK-293 cells (control), or after transfection of LMX1A siRNA (#144), 200 ng/well constitutively active S33Y-b-catenin and LEF1 cDNA alone or together, pMES-Lmx1a (encoding the full-length mouse LMX1A protein) or non-targeting (nt) siRNA (control), for the detection of LMX1A transcription in these cells. Densitometric analyses (normalized to Gapdh mRNA levels, not shown) revealed an approx. 50% reduction of endogenous LMX1A mRNA levels in LMX1A siRNA-transfected HEK-293 cells, and an approx. 300% increase of LMX1A transcripts in these cells after transfection of pMES-Lmx1a. All other conditions had no detectable influence on endogenous LMX1A transcript levels in HEK-293 cells. (B) Fold change of luciferase (LUC) activity in HEK-293 cells (relative to Pitx3 promoter/reporter only-transfected cells, set as 1; white bar) after co-transfection with 166 ng/well Lmx1a, 200 ng/well S33Y-b-catenin, 200 ng/well LEF1, and LMX1A siRNA (#144) alone or together in different constellations, as indicated below the graph [n = 3 independent experiments; statistical testing for significance was done using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc tests; F(6,14) = 10.92, P < 0.0001]. *P < 0.05; ns, not significant.