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

FIGURE 7.

FIGURE 7.

Oxidant signaling contributes to AMPK activation upon matrix deprivation. A, cells were loaded with DCFDA, a fluorescent dye for ROS measurements. Parallel wells were loaded with calcein AM as control. Fluorescence was measured at excitation 490/emission 520 nm in a spectrofluorometer in a plate reader format as a time course as represented in the graph. The 1st black arrow represents the point of mechanical detachment in the time course. The 2nd black arrow represents the addition of positive control H2O2. Fluorescence values were normalized to initial reading. DCFDA fluorescence/calcein fluorescence was calculated at each time point. Att, attached; Det, detached. MDA-MB 231 cells (B) G361 cells (C) were loaded with DCFDA, and ROS levels were measured as described above. The black arrow represents the point of mechanical detachment in the time course (n = 4 with three technical repeats each). Values are expressed as mean ± S.E. D, MDA-MB 231 cells pretreated with either vehicle control or MCI-186 (200 μm), for 2 h, were cultured under attached conditions or detached for 10 min, and Western blotting was performed to measure the levels of pAMPKα and AMPK (n = 6). The scatterplot depicts fold change in pAMPKα/tubulin ratio; ***, p < 0.001. Error bars represent ± S.E. E, MDA-MB 231 cells pretreated with either vehicle control or MCI-186 (200 μm), for 2 h, were detached for 10 min, and Western blotting was performed to measure the levels of pLKB1 Ser-428 and LKB1 (n = 5). The scatterplot depicts fold change in pLKB1/tubulin ratio; **, p < 0.01. Error bars represent ± S.E. F, MDA-MB 231 cells pretreated with either vehicle control or 1 mm NAC, for 2 h, were then either allowed to remain attached or detached for 10 min, and Western blotting was performed to measure the levels of pAMPKα and AMPK (n = 4). The scatterplot depicts fold change in pAMPKα/tubulin ratio; **, p < 0.01. Error bars represent ± S.E. G, LKB1-deficient G361 cells pretreated with either vehicle control or MCI-186 (200 μm), for 2 h, were then either allowed to remain attached or detached for 10 min, and Western blotting was performed to measure the levels of pAMPKα and AMPK (n = 4). The scatterplot depicts fold change in pAMPKα/tubulin ratio; **, p < 0.01. Error bars represent ± S.E.