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. 2014 Mar 14;21(8):1185–1197. doi: 10.1038/cdd.2014.34

Figure 5.

Figure 5

K-RasV12-induced activation of p38 is required for ROS generation and consequent malignant transformation. (a) Western blot for activation status of ERK, p38, and JNK MAPK in K-RasV12-transduced cells and control vector MFG-transduced cells. (b and c) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with DN-ERK, DN-p38, or DN-JNK (b) or treatment with SB203580, inhibitor specific to p38 MAPK (c), and are subsequently transduced with K-RasV12. (d) Soft agar colony formation in control cells or cells that are transfected with DN-ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-RasV12. (e) Tumor growth curves of xenografts derived from parental Rat2 cells, K-RasV12-transduced cells, or K-RasV12-transduced cells that are transfected with DN-p38. (f) Western blot for the phosphorylation status on Thr505 of PKCδ in Rat2 cells that are transfected with DN-p38 or control pcDNA, and are subsequently transduced with K-RasV12 or control vector MFG. (g) Western blot for the analysis of interaction between PKCδ and p47phox after GST-p47phox pull-down and immunoprecipitation with anti-PKCδ in cells that are transfected with DN-ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-RasV12. (h) Western blot for phosphorylation status on serine residues of p47phox after immunoprecipitation with anti-phospho-serine antibody, or membrane translocation of p47phox in cells that are transfected with DN ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-RasV12. (i) Western blot for the analysis of interaction between PKCδ and p47phox after co-immunoprecipitation with anti-p47phox or anti-PKCδ antibody in cells that are transfected with WT p38 or control pcDNA, and are subsequently transduced with K-RasV12 or control vector MFG. (j) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with WT p38 or control pcDNA, and are subsequently transduced with K-RasV12 or control vector MFG. WT, wild type; DN, dominant-negative mutant; Error bars represent mean±S.D. of triplicate samples. *P<0.001