AMPK Methods and Protocols

B C

A

-Glu Baf. A1

-Glu Baf. A1

-Glu

Baf. A1

-Glu

Baf. A1

siControl

siAMPK

ULK1

135 P-ULK1 (Ser555)

135

siControl

−

−+

−

−+

++−

−+

−

−+

++

-Glu

Baf. A1

siAMPK

AMPK

P-AMPK (Thr172)

LC3-I

p62

actin

LC3-II

63

63

63

48

17

ACC

245 P-ACC (Ser79)

245

Raptor

(^180) P-Raptor (Ser792)
180
LC3-II/actin: 7.75
1.61 0.98
1.00
P-ULK1/ULK1: 1.00

• -Glu
  Baf. A1
  -Glu & Baf. A1

0

30

60

90

120

150

LC3 dots per cell

siControl

siAMPK

**

***

**

n.s.

1.040.61

19.9630.770.850.73

1.530.95

8.15

0.82

7.59

Fig. 2Assessment of the AMPK dependency of autophagy using siRNA-mediated knockdown. (a) Represen-
tative confocal images of U2OS cells, which were transfected with the indicated siRNA pools and cultured in
glucose-free medium (Glu). Scale bar, 20μm. Bafilomycin A1 (Baf. A1) was added to the cells to check for
an intact autophagic flux. Endogenous LC3 was used to monitor autophagy through indirect immunofluores-
cence microscopy. (b) The number of endogenous LC3 dots within the cells treated as in (a) was counted for
each treatment (n>100). The shown results are mean valuesSD. The significance was tested by applying
a Student’st-test (n.s.not significant, **p0.05, ***p0.01). The highest number of LC3 dots in the sample
treated with glucose deprivation (-Glu) and Baf. A1 in comparison to the single treated samples indicates an
intact autophagic flux after glucose deprivation in U2OS cells. (c) U2OS cells were transfected and treated as
in (a). Efficiency of the AMPK knockdown was shown by immunoblot analysis. The AMPK knockdown leads to
a reduced substrate phosphorylation and autophagy induction after glucose deprivation. A reduced autophagy
flux is observed in the AMPK knockdown cells compared to the cells treated with the control siRNA, because
the AMPK knockdown cells accumulate LC3-II in the combined -Glu and Baf. A1 treatment to a lesser degree
than the control siRNA-treated cells

Autophagy 381