Figure 5.
AMPK is activated by ETC inhibition. (A) Representative western blot of cortical neuron lysates was probed with an antibody specific for phospho-threonine 172 of AMPK and with anti-GAPDH. On a separate blot, the same lysates were probed with anti-AMPK and anti-GAPDH. Cultures were treated with ETC inhibitors for the indicated times and concentrations. (B) Quantification of replicate blots from cultures treated as described in A. n = lysates from three independent animals. (C) Quantification of mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control, as well as mito-dsRED and meGFP. n = 7–10 axons per treatment from five independent animals. (D) Quantification of mitochondrial motility in hippocampal cultures expressing dominant negative AMPK (DN-AMPK) or empty vector control, as well as mito-dsRED and meGFP. DIV8–10 cultures were treated with 4 nM AntA or vehicle for 1 h and then imaged. n = 7–10 axons per treatment from five independent animals. (E) Quantification of the effects of a 4-day AMPKα1 knockdown on mitochondria-associated actin. Fibroblasts were cultured in galactose media to increase dependence on ETC function and then treated for 1 h with 40 nM AntA or vehicle. F-actin and mitochondria were visualized by live imaging using GFP-F-tractin and mito-mRaspberry and expressed either control or AMPKα1 shRNAs. shRNA constructs also expressed a nuclear-targeted iRFP (NLS-iRFP), and only cells expressing the NLS-iRFP signal were analyzed. n = 10–14 cells were imaged from 5 independent biological repeats. For all graphs, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P value for B was calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction. The P value for C was calculated by performing a two-tailed, unpaired t test with Welch’s correction. A blocked ANOVA was performed with Tukey’s multiple comparison correction for D and E. Only P values ≤0.05 are shown for D, and select comparisons are shown for E. Source data are available for this figure: SourceData F5.

AMPK is activated by ETC inhibition. (A) Representative western blot of cortical neuron lysates was probed with an antibody specific for phospho-threonine 172 of AMPK and with anti-GAPDH. On a separate blot, the same lysates were probed with anti-AMPK and anti-GAPDH. Cultures were treated with ETC inhibitors for the indicated times and concentrations. (B) Quantification of replicate blots from cultures treated as described in A. n = lysates from three independent animals. (C) Quantification of mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control, as well as mito-dsRED and meGFP. n = 7–10 axons per treatment from five independent animals. (D) Quantification of mitochondrial motility in hippocampal cultures expressing dominant negative AMPK (DN-AMPK) or empty vector control, as well as mito-dsRED and meGFP. DIV8–10 cultures were treated with 4 nM AntA or vehicle for 1 h and then imaged. n = 7–10 axons per treatment from five independent animals. (E) Quantification of the effects of a 4-day AMPKα1 knockdown on mitochondria-associated actin. Fibroblasts were cultured in galactose media to increase dependence on ETC function and then treated for 1 h with 40 nM AntA or vehicle. F-actin and mitochondria were visualized by live imaging using GFP-F-tractin and mito-mRaspberry and expressed either control or AMPKα1 shRNAs. shRNA constructs also expressed a nuclear-targeted iRFP (NLS-iRFP), and only cells expressing the NLS-iRFP signal were analyzed. n = 10–14 cells were imaged from 5 independent biological repeats. For all graphs, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P value for B was calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction. The P value for C was calculated by performing a two-tailed, unpaired t test with Welch’s correction. A blocked ANOVA was performed with Tukey’s multiple comparison correction for D and E. Only P values ≤0.05 are shown for D, and select comparisons are shown for E. Source data are available for this figure: SourceData F5.

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