Fig 1.
AMPK activation and mass spectrometry analysis of cell surface proteins.
(A) RPE cells were stimulated with 100 μM A-769662 in media containing 0.1% FBS for indicated times. Shown are representative immunoblots using antibodies as indicated. (B) Shown is a diagram depicting cell stimulation, surface biotinylation, purification of biotinylated proteins, mass spectrometry and peptide identification. We thus identified a total of 838 proteins within all cell surface fractions, of which 653 exhibited reduced detection in the cell surface fraction of cells treated with A-769662, 93 proteins exhibited increased cell surface abundance in cells treated with A-769662, and a further 92 were classified as exhibiting largely unaltered detection in the cell surface fraction upon AMPK activation. A complete list of identified proteins can be found in S1 Table.
Table 1.
Gene Ontology terms depleted from the cell surface upon A-769662 treatment.
Table 2.
Proteins with cell adhesion and migration GO classification depleted from the cell surface upon A-769662 treatment.
Fig 2.
STRING analysis reveals functional interaction of cell migration and adhesion proteins depleted from the cell surface by A-769662 treatment.
The list of 32 proteins with cell adhesion and migration GO annotation (Table 2) was subjected to STRING analysis to visualize known and predicted interactions [54]. Shown is a graphical representation of the output of this analysis. Highlighted is the predicted interaction of proteins identified as depleted from the cell surface fraction upon A-769662 treatment with β1-integrin (ITGB1).
Fig 3.
Treatment with A-769662 reduces cell migration.
RPE cells were subjected to an epithelial wounding cell migration assay, either under conditions of continuous stimulation with 100 μM A-769662 or unstimulated (control). (A) Shown are representative micrographs of cells immediately after wounding (0 h) or 24 h after wounding, as indicated, with the region of the wound indicated by dashed white lines. (B) The coverage of the wounded area by cells 24 hours after wounding was quantified; shown are the means ± SE of the percent of wounded area covered by migrating cells in 24h in control and A-769662 treated cells (n = 3, p < 0.05).
Fig 4.
Treatment with A-769662 reduces cell surface β1-integrin levels.
(A) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (B) Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified as described in Materials and Methods and S5B Fig. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 4 independent experiments). (C) RPE cells were stimulated with 2 mM AICAR for 90 min or left unstimulated (basal), followed by cell-surface biotinylation, purification of biotinylated proteins and immunoblotting of fractions with an antibody specific to β1-integrin. Shown is an immunoblot of cell surface β1-intergin (top panel, corresponding to the streptavidin pull-down), and of the corresponding intracellular β1-integrin (bottom panel, corresponding to the above supernatant), representative of 4 independent experiments. (D) Shown are representative immunoblots of whole-cell lysates prepared from cells stimulated with either 100 μM A-769662, 2 mM AICAR, 40 μM compound C (each for 90 min) or left unstimulated (control), probed with antibodies to detect total cellular β1-integrin or actin (load).
Fig 5.
Inhibition of AMPK by siRNA gene silencing or by compound C prevents the reduction in cell surface β1-integrin elicited by A-769662 treatment.
(A-C) RPE cells were transfected with siRNA targeting AMPK α1/2 or non-targeting (NT, control) siRNA. (A) Whole cell lysates were prepared and resolved by immunoblotting and probed with anti-AMPK α1/2 or anti-actin antibodies. Shown are immunoblots representative of at least 3 independent experiments. (B) Following siRNA transfection, cells were treated with 100 μM A-769662 for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (C) Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments). (D) RPE cells were treated with 100 μM A-769662 or 40 μM compound C, alone or in combination, for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (E) Cell surface β1-integrin levels obtained by fluorescence microscopy as in (D) were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).
Fig 6.
Treatment with A-769662 does not change cell surface TfR levels.
(A) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on Transferrin Receptor (TfR). Shown are representative fluorescence micrographs depicting cell surface TfR fluorescence. Scale = 5 μm (B) Cell surface TfR levels obtained by fluorescence microscopy were quantified. Shown are the cell surface TfR measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).