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. 2016 May 11;291(28):14410–14429. doi: 10.1074/jbc.M116.731257

FIGURE 3.

FIGURE 3.

Role of upstream kinases in detachment-induced AMPK activation in LKB1 containing MDA-MB 231 cells. A, MDA-MB 231 cells stably expressing pGIPZ non-targeting shRNA (NT) or shRNA targeting LKB1 (shLKB1) or shRNA targeting CaMKKβ (shCaMKKβ) were harvested for immunoblotting with LKB1 and CaMKKβ (n = 5). Numbers indicate relative LKB1/tubulin and CaMKKβ/tubulin ratio. B, MDA-MB 231 stably expressing NT, shLKB1, or shCaMKK were cultured under attached conditions. Levels of pAMPKα, pACC, AMPK, and ACC were measured by Western blotting (n = 3). The numbers depict relative pAMPKα/AMPK ratio. pAMPKα blot was obtained at high exposure (H.E). C, MDA-MB 231 cells expressing NT, shLKB1, or shCaMKKβ were cultured under attached (Att) and detached (Det) conditions. pAMPKα and pACC were measured by Western blotting (n = 7–8). Numbers indicate relative pAMPKα/tubulin ratio. The scatterplot depicts the fold change in pAMPKα/tubulin ratio of the knockdown cells relative to control NT cells; ***, p < 0.001. Error bars represent ± S.E. D, MDA-MB 231 cells were pretreated with STO-609, a specific inhibitor for CaMKKβ, for 2 h. Cells were then cultured under attached and detached (10 min) conditions, and pAMPKα, pACC, total AMPK, and ACC levels were measured by Western blotting (n = 6). The scatterplot depicts the fold change in pAMPKα/tubulin ratio; ***, p < 0.001. Error bars represent ± S.E. ns, non-significant. E, MDA-MB 231 cells stably expressing NT, shLKB1, or shCaMKKβ were cultured under attached and detached conditions for 24 h. pAMPKα and pACC were measured by Western blotting (n = 3). The numbers represent pAMPKα/tubulin ratio. F, MDA-MB 231 and MCF7 were cultured under attached and detached conditions. pLKB1 (Ser-428) and LKB1 levels were measured by Western blotting (n = 4). Scatterplot depict relative fold change in pLKB1/LKB1 ratio. Error bars represent ± S.E. G, MCF7 cells were cultured under attached and detached conditions, and immunocytochemistry was performed for LKB1. Representative confocal images of equatorial sections are shown. AU, arbitrary units.