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Fig. 4. Suppression of p38
by siRNA treatment or expression of dominant-negative mutant of p38 blocks canonical Wnt–β-catenin–Lef/Tcf pathway. (A) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse p38 or JNK for 48 hours, and the lysates were assayed for cytosolic β-catenin levels. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin); immunoblots probed with anti-actin antibody (actin) were used as loading controls. (B) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse p38 for 48 hours followed by stimulation with Wnt3a for 7 hours. Lef/Tcf-sensitive transcription was determined. The data represent mean values ± s.e.m. from a single experiment performed in triplicate and is representative of three separate experiments whose results were in high agreement. (C) Rescue experiment performed by transfection of hp38 into F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter in which p38 MAPK was knocked down by siRNA treatment. Lef/Tcf-sensitive transcription was determined. The data represents mean values ± s.e.m. from a single experiment performed in triplicate and is representative of three separate experiments whose results were in high agreement. (D<E) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were either transfected with empty vector (–) or with indicated amounts of Flag-tagged dominant-negative mutant (DN) of p38 MAPK [p38 (AGF)] for 24 hours and the lysates were assayed either for β-catenin stabilization after 4 hours of Wnt3a treatment (D) or luciferase reporter activity after 7 hours of Wnt3a treatment (E). Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), anti-p38 antibody (p38) and the immunoblots with anti-actin antibody (actin) were used as loading controls. (F,G) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either 100 nM siRNA specific to mouse p38 or dominant-negative mutant (DN) of p38 MAPK for 48 hours. The cells were then treated with SB203580 (6 µM) for 1 hour followed by stimulation with Wnt3a for 7 hours. Lef/Tcf-sensitive transcription (F) and p38 MAPK activation (G) was determined as described in Materials and Methods. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.
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