Rat Rem2 shRNAs did not change VGCC peak currents in hippocampal neurons.

<p><i>A,</i> VGCC currents from control neurons and neurons transfected with rRem2-targeted shRNAs. There was no change in current density at either 4 d post-transfection (N = 11, p = 0.24) or 10 d post-transfection (N = 5–7, p = 0.48). <i>B,</i> Rem2 mRNA relative values in hippocampal neuron culture was measured at 1, 4, 8 and 14 DIV (N = 5–9) with PCR quantitative analysis. The relative value was normalized to the value of 1 DIV.</p

shRNA did not reduce endogenous Rem2 measured by immunocytochemistry in hippocampal neurons.

<p><i>A</i> and <i>C,</i> Exemplar images of neurons transfected with GFP or GFP-rRem2; or co-transfected with GFP and control vector <i>versus</i> GFP and a pool of rRem2 shRNAs. Left column, expressed GFP; right column, rRem2 detected with antibody against Rem2 and visualized with secondary antibody Cy3. Scale bar, 20 µm. <i>B</i> and <i>D,</i> Summarized GFP and Cy3 fluorescent levels indicated with mean gray value. Neurons overexpressing rRem2 (n = 41) showed significantly stronger fluorescent signal than control neurons (N = 15) (p = 0.0003). There was not significantly difference between neurons transfected with rRem2 shRNA (N = 51) and control plasmid (N = 48) (p = 0.25).</p

Rat Rem2 shRNAs reduced mEPSCs frequency in hippocampal neurons.

<p><i>A,</i> Exemplar recording of mEPSCs from neurons transfected with rRem2-targeted shRNAs or the control plasmid. <i>B,</i> and <i>C,</i> Summarized mEPSCs frequency and amplitude 4 (N = 6) and 10 d (N = 12) after transfection, showing that the mEPSCs frequency was reduced significantly at 10 d after transfection of shRNAs compared with control transfection (p = 0.006). There was no change in amplitude (p = 0.55).</p

GFP-hRem2, which was insensitive to rRem2-targeted shRNAs, could not rescue the reduction in mEPSCs frequency induced by shRNAs.

<p><i>A,</i> and <i>B</i>, immunoblot for GFP of lysates of untransfected HEK cells or cells transfected with GFP, GFP-rRem2 plus either rRem2 shRNAs or empty vector, and GFP-hRem2 plus either rRem2 shRNAs or empty vector. The rRem2-targeted shRNAs reduced GFP-rRem2 expression (N = 4, p = 0.03). <i>C,</i> VGCC currents recorded from cultured hippocampal neurons 3 d after transfection with GFP-hRem2 (N = 8) or GFP (N = 7). GFP-hRem2 reduced VGCC currents (p = 0.03). <i>D,</i> mEPSC frequency recorded at 4 d (N = 8) and 10 d (N = 16–21) after transfection with empty vector, rRem2-targeted shRNAs, or rRem2-targeted shRNAs together with GFP-hRem2. Transfection with rRem2-targeted shRNAs reduced mEPSCs frequency at 10 d compared to control (p = 0.02). Co-transfection with GFP-hRem2 did not rescue the effect of rRem2-targeted shRNAs (p = 0.12).</p

Over-expression of Rem2 reduced VGCCs without changes in mEPSCs in cultured hippocampal neurons.

<p><i>A,</i> Exemplar VGCCs were recorded from neurons transfected with GFP-rRem2 or GFP. The currents were induced by a ramp protocol −80 mV to +50 mV over 500 ms after a 50 ms step to −80 mV from a holding potential of −70 mV. <i>B,</i> Summarized VGCC currents recorded 4 (N = 7) and 10 d (N = 8) after Rem2 over-expression, showing VGCCs decreased markedly at 4 d (p = 0.03) and there was not significant difference at 10 d (p = 0.1). <i>C,</i> Exemplar recording of mEPSCs from neurons transfected with GFP-rRem2 or GFP only. <i>D,</i> and <i>E</i>, mEPSCs showed no change in mEPSCs frequency or amplitude at 4 d post-transfection (N = 9–10; frequency p = 0.11; amplitude p = 0.53) or 10 d post-transfection (N = 15; frequency p = 0.19; amplitude p = 0.36).</p

Raptor phosphorylation by AMPK is required for AICAR-induced autophagy in TSC2^−/− MEF cells.

<p>(A) TSC2^−/−, p53^−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm. (B) TSC2^−/−, p53^−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1. (C) Schematic representation of AMPK-mediated suppression of the inhibitory effect of mTORC1 on the ULK1 autophagic complex.</p

The AMPK-ULK1 interaction is important for ULK1-mediated autophagy.

<p>(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654–828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. (B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654–828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm. (C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81). (D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554–828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's <i>t</i>-test and the asterisks indicate <i>P</i><0.0001.</p

AMPK interacts with ULK1 directly.

<p>(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.</p

AICAR induces AMPK phosphorylation of raptor and the recruitment of 14-3-3 to the ULK1-mTORC1 complex.

<p>293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.</p

AMPK and mTORC1 interact with ULK1 through the PS domain and the kinase domain of ULK1, respectively.

<p>(A) A schematic of ULK1 illustrating its kinase domain, proline/serine-rich domain, C-terminal (CT) domain, and regions for binding to AMPK and mTORC1. (B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.</p