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. 2012 Jan 1;125(1):59–66. doi: 10.1242/jcs.085803

Fig. 5.

Fig. 5.

p38MAPK is involved in Scribble-knockdown-mediated cell competition. (A) Effect of the p38 MAPK inhibitor SB202190 on death of MDCK-pTR Scribble shRNA cells surrounded by normal MDCK cells. MDCK-pTR Scribble shRNA cells were fluorescently labeled with CMFDA dye and mixed with normal MDCK cells at a ratio of 1:10, and cultured with tetracycline for 64 hours in the absence or presence of SB202190. SB202190 was added during the final 40 hours. Cells were incubated with SYTOX Blue to label dead cells. Results represent the means ± s.d. *P<0.05; n=172 and 170 from four independent experiments. (B) Establishment of MDCK-pTR Scribble shRNA cells stably expressing dominant-negative GFP–p38-MAPKα (p38DN) in a tetracycline-inducible manner. MDCK cells or MDCK-pTR Scribble shRNA + GFP–p38DN cells were incubated in the presence or absence of tetracycline for 64 hours. Cell lysates were examined by western blotting using anti-Scribble, anti-GFP, anti-p38-MAPKα or anti-GAPDH antibody. Arrows and arrowhead indicate the position of GFP–p38DN and endogenous p38MAPK proteins, respectively. It should be noted that expression of endogenous p38 MAPK is downregulated upon expression of exogenous GFP-p38DN. (C) Expression of dominant-negative p38MAPKα (p38DN) in Scribble shRNA cells rescues the cell competition phenotype. MDCK-pTR Scribble shRNA cells or MDCK-pTR Scribble shRNA + GFP–p38DN cells were fluorescently labeled with CMFDA dye and mixed with normal MDCK cells at a ratio of 1:10, and incubated in the presence of tetracycline for 64 hours. Results are expressed relative to the value for MDCK-pTR Scribble shRNA cells, and represent the mean ± s.d. **P<0.05, n=380 and 312 from three independent experiments. (D) Effect of the p38 MAPK inhibitor SB202190 on active Bak immunostaining in MDCK-pTR Scribble shRNA cells surrounded by normal MDCK cells. MDCK-pTR Scribble shRNA cells were fluorescently labeled with CMFDA dye and mixed with normal MDCK cells at a ratio of 1:10. Cells were incubated in the presence or absence of tetracycline for 64 hours with the addition of 30 μM blebbistatin for the final 24 hours to inhibit extrusion of apoptotic cells. SB202190 was added during the final 24 hours. Results represent the means ± s.d. *P<0.05; n=292, 265 and 267 from three independent experiments.