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. 2010 Mar 23;123(8):1352–1362. doi: 10.1242/jcs.056176

Fig. 4.

Fig. 4.

KSRP negatively regulates Wnt/β-catenin signaling. (A) F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific to Khsrp, Dvl2 or Dvl3 (100 nM) for 48 hours and the Ctnnb1 and Ppia (cyclophilin A) mRNA levels were quantified using quantitative PCR. The data represent Ctnnb1 mRNA normalized to the Ppia (cyclophilin A) mRNA levels (mean values ± s.e.m.) from three independent experiments whose results were in high agreement. F9 cells were treated with either control siRNAs (100 nM) or siRNAs specific for mouse Khsrp (100 nM) for 48 hours and the lysates were assayed either for cytosolic β-catenin levels (B) or Lef/Tcf-sensitive transcription (C). Upper panel displays mean values ± s.e.m. from three independent experiments; the lower panel displays representative blots. (D) F9 cells were treated with control siRNAs (100 nM) or siRNAs specific for mouse Khsrp (100 nM) for 48 hours before stimulation with Wnt3a for 4 days. Subsequently, formation of primitive endoderm was assayed by immunostaining the cells with anti-cytokeratin endo A antibody (TROMA-1), a hallmark of PE formation. Typical phase-contrast images (PC) and the indirect immunofluorescence images (IIF) are shown from a single experiment, representative of two independent experiments. F9 cells (E) or HEK293 cells (F) were transfected with FLAG-KSRP for 24 hours and the lysates were assayed for cytosolic β-catenin stabilization. (G) F9 cells were transfected with indicated amounts of FLAG-KSRP for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription after stimulation with Wnt3a for 7 hours. (H) F9 cells were transfected with HA-Dvl3-GFP2 alone or together with increasing amounts of FLAG-KSRP for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription. (I) F9 cells were transfected with indicated amounts of an unrelated protein, mouse S-Phase kinase associated protein 1A (mSKP1A) for 24 hours and the lysates were assayed for Lef/Tcf-sensitive transcription. The data represents mean values ± s.e.m. from three independent experiments *P<0.05; **P<0.01 versus control (−Wnt3a). #P<0.05; ##P<0.01 versus control (+Wnt3a).