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. Author manuscript; available in PMC: 2017 Apr 13.
Published in final edited form as: Mol Cancer Res. 2016 May 2;14(8):683–695. doi: 10.1158/1541-7786.MCR-15-0479

Figure 3. Effect of expression of constitutively active CAMKK2 on AMPK activation and the cell cycle in G361 cells.

Figure 3

(A) Cells were left untransfected, or were transfected with DNAs encoding GFP fused to full length (1-588, GFP-WT), or truncated (1-471, GFP-CA) CAMKK2. After 36 hr, AMPK activity was measured in immunoprecipitates; significant differences by 1-way ANOVA compared with untransfected control (***p<0.001) or GFP-WT (††p<0.01) are shown. (B) Analysis by Western blotting of extracts of untransfected cells and cells transfected with DNAs encoding GFP-WT or GFP-CA. Extracts were made after 36 hr and antibodies used were anti-GFP (top panel), anti-pt172, anti-AMPK-α or anti-actin. Results show samples from duplicate cell incubations: similar results were obtained in three independent experiments. (C) AMPK activity in immunoprecipitates from cells 36 hr after transfection with DNAs encoding GFP-WT or GFP-CA, or untransfected controls. Cells were then incubated with or without 10 μM A23187 for 1 hr; results are mean ± S.D. (n = 2); significant effects by two way ANOVA compared with vehicle controls, **p<0.01, or untransfected controls (††††p<0.0001, ††p<0.01) are shown. (D) Analysis by Western blotting of extracts of the same cells shown in (C). (E) Percentage of cells in G1, S or G2/M for cells transfected with DNAs encoding GFP, GFP-WT or GFP-CA (mean ± S.D., n = 3). After 36 hr, cells were treated with nocodazole (70 ng.ml-1) and 18 hrs later were fixed, stained with propidium iodide and the DNA content analyzed by flow cytometry. The flow cytometer was set-up to analyze only cells expressing GFP. Significant differences between the percentage of that cell cycle phase compared with GFP alone (****p<0.0001) or between GFP-WT and GFP-CA (††††p<0.0001) are shown (2-way ANOVA). Similar results were obtained in three identical experiments. (F) As (E), except that cells were transfected with DNAs encoding GFP, GFP-CA, or GFP-LKB1 plus STRADA and CAB39. Similar results were obtained in two identical experiments.