Data in support of 5′AMP-activated protein kinase alpha regulates stress granule biogenesis

This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5′-AMP-activated kinase (AMPK). Our results verify the specific association of AMPK-α2, but not AMPK-α1, with SGs. We also provide validation data for the isoform-specific recruitment of the AMPK-α subunit to SGs using (i) different antibodies and (ii) a distinct cellular model system. In addition, we assess the SG association of the regulatory AMPK β- and γ-subunits. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in “5′AMP-activated protein kinase alpha regulates stress granule biogenesis” [1].


a b s t r a c t
This data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5 0 -AMP-activated kinase (AMPK). Our results verify the specific association of AMPK-α2, but not AMPK-α1, with SGs. We also provide validation data for the isoform-specific recruitment of the AMPK-α subunit to SGs using (i) different antibodies and (ii) a distinct cellular model system. In addition, we assess the SG association of the regulatory AMPK βand γ-subunits. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in "5 0 AMP-activated protein kinase alpha regulates stress granule biogenesis" [1]. The recruitment of the regulatory AMPK-β and AMPK-γ subunits to SGs under different stress conditions is assessed.
Data on the impact of AMPK knockdown on the abundance of core SG proteins are provided.

Data, materials and methods
AMPK serves as a master metabolic regulator in eukaryotic cells [2]. Moreover, AMPK is also critical to other cellular functions, such as the stress response [3]. The formation of cytoplasmic SGs is one of the hallmark responses to many types of stress [4]. Here, we used different antibodies (Fig. 1) and a distinct cell line from another organism (Fig. 2) to verify the isoform-specific recruitment of the AMPK-α subunit to SGs. In addition to the catalytic α subunits, we assessed the interaction of regulatory β and γ AMPK subunits with SGs upon treatment with arsenite or diethyl maleate (DEM), components that induce oxidative stress (Figs. [3][4][5]. AMPK-α knockdown changed SG parameters [1], and we tested whether this can be linked to the abundance of SG marker proteins TIA-1/TIAR, G3BP1 and HuR. To this end, we quantified the levels of core SG proteins under control and AMPK-α knockdown conditions. The knockdown of AMPK-α1 or α2 did not cause significant changes in the concentration of these three SG marker proteins (Fig. 6).

Cell culture, stress and drug treatment
HeLa cells were grown in Dulbecco's modified eagle medium (DMEM) containing antibiotics and 8% Fetal Bovine Serum (FBS). Mouse Embryonic Fibroblasts were cultured in DMEM, supplemented Fig. 1. Isoform-specific recruitment of AMPK-α to SGs. The isoform-specific association of the catalytic subunit with SGs was verified in HeLa cells with a different set of primary antibodies against AMPK-α1 or AMPK-α2 (Upstate, see Section 1). Consistent with results in our main paper [1], these unrelated isoform-specific antibodies located AMPK-α2 to DEM-induced SGs, which were demarcated with the SG marker G3BP1. Scale bar is 20 μm. with l-glutamine and 8% FBS. Cultures were maintained in a 37 1C incubator containing 5% CO 2 . To induce oxidative stress, cells were incubated with 0.5 mM sodium arsenite or sterile water (control) for 1 h. Alternatively, HeLa cells were incubated with 2 mM diethyl maleate (DEM) or ethanol (control) for 4 h.

Cell extracts
HeLa cell proteins were solubilized in gel sample buffer as described [7]. Samples were incubated at 95 1C for 10 min and vortexed with silica beads to shear DNA. Proteins were precipitated with trichloroacetic acid for 20 min on ice and collected by centrifugation (microfuge, 1 min at 13,000 rpm). Sediments were resuspended in gel sample buffer and analyzed by Western blotting.