AMPK Methods and Protocols

2. Seed 1.2 106 cells, counted using a cell counter, onto a 10 cm
   dish containing a glass coverslip to be able to perform immu-
   noblot and indirect immunofluorescence analysis from the
   same experiment as described in Subheadings3.5 and 3.6 (see
   Notes 13– 15 ). Grow cells under normal growth conditions at
   37 C with 5% CO 2 to reattach overnight (seeNote 16).


3. To induce AMPK-dependent autophagy, stimulate the cells
   either with 1 mM AICAR and 50μM A-769662 (AþA) for
   16 h (seeNote 17), or after a wash with pre-warmed PBS,
   replace the full medium with pre-warmed, glucose-free
   DMEM for 16 h (seeFig. 1)(seeNotes 18and 19 ). Afterward
   lyse or fixate the cells for further analysis (seeSubheadings3.4
   and 3.6)

3.2 Analysis of the
Autophagic Flux

A variety of methods can be employed to assess autophagic flux

since steady-state analysis alone (seeSubheading3.1,step 3) can

lead to misinterpretations. Two approaches are described in the

following:

1. Use Baf. A1, which prevents the fusion of the autophagosome
   with the lysosome (seeFigs.2 and 3). Add Baf. A1 to the cells
   4 h prior to lysis at a final concentration of 200 nM (seeNotes
   17 and 20 ).


2. A double-tagged mCherry-EGFP-LC3 fusion protein allows
   the differentiation between autophagosomes and autolyso-
   somes and an estimation of their ratio (seeFig. 3b)(seeNote
   21 ). Seed and stimulate the NIH3T3 cells stably expressing the
   double-tagged mCherry-EGFP-LC3 construct as described in
   Subheading3.1 and instep 1of this section (seeNote 16).

3.3 siRNA
Transfection

The siRNA-mediated knockdown of AMPKα1/2 or AMPKα1/

2 knockout cells can be employed to demonstrate AMPK depen-

dency of the induction of autophagy (seeFig. 2). In the following

we describe the protocol for using the siRNA-mediated

knockdown.

1. Seed 4 105 cells onto a 6 cm dish containing a glass coverslip
   (seeNotes 13, 14 , 15 , and 22 ). Grow cells under normal
   growth conditions at 37C with 5% CO 2 for about 2 h until
   the transfection (seeNote 23).


2. For transfection, incubate 12μl of the indicated 20μM siRNA
   oligo pools with 250μl of Opti-MEM®and 20μl of HiPerFect
   transfection reagent for 10 min at room temperature. After-
   ward add the mixture to the cells dropwise resulting in a final
   concentration of 50 nM siRNA oligos in the medium.


3. Thereafter incubate the cells under normal growth conditions
   at 37C with 5% CO 2 for 72 h to allow uptake of the siRNA-
   containing liposomes (seeNote 24).

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