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. 2021 Feb 15;131(4):e135465. doi: 10.1172/JCI135465

Figure 7. Concerted Activation of AKT and GATA2/AR signaling is essential for mediating MAPK4 tumor-promoting activity in PCa.

Figure 7

(A) Western blots on LAPC4 cells and VCaP cells with knockdown of MAPK4 (G2, G4) or control (NT). (B) Western blots, (C) qPCR, (D) Proliferation assays, and (E) Soft-agar assays (original magnification: × 50) on the engineered LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4 (iWT), MAPK4S186A (iS186A), MAPK4D254A (iD254A), or control (iCtrl). Data represent mean ± SEM. Adjusted P values determined by 1-way ANOVA followed by Dunnett’s multiple comparisons. ***P ≤ 0.001. ****P ≤ 0.0001. (F) Proliferation assays and Western blots on the LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4 (iWT), MAPK4D254A (iD254A), or control (iCtrl), treated with 1 μM of AKT inhibitor MK2206 or DMSO control. (G) Proliferation assays and Western blots on the LNCaP cells with 0.5 μg/mL Dox-induced expression of MAPK4, MAPK4D254A, or control, also infected with lentivirus expressing AKT1-DD mutant or control. (H) Proliferation assays on the LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4, MAPK4D254A, GATA2, or control, also overexpressing AKT1-DD or control. The growth of these cells in 10% FBS, 5% CSS, and 5% CSS plus 1 nM R1881 were compared. Also shown are Western blots on these cells cultured in 10% FBS. (I) Proliferation assays and Western blots on the control (NT) or MAPK4-knockdown (G2) VCaP cells with 0.5 μg/mL Dox-induced expression of GATA2 (iGATA2), AKT1-DD (iAKT1DD), or control (iCtrl). Data are representative of at least 3 independent experiments.