Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a , b ) Starvation induces a stronger autophagic response than rapamycin. In a , MEFs stably expressing GFP-LC3 were incubated in starvation media or treated with 1-μM rapamycin for the indicated time. Representative images from three independent experiments shown, average number of puncta per cell was quantitated and expressed as fold change relative to 0 h (fold change ±s.d., n =30. two-tail Student's t -test, * P <0.05). In b , MEFs were lysed at the indicated time points and immunoblotted for endogenous LC3 and other proteins as indicated. ( c ) Kinetics of mTOR substrate dephosphorylation in response to starvation or pharmacological inhibition of mTOR. MEFs were incubated in starvation media or media containing 1 μM of either Torin1 or rapamycin, and lysed at the indicated time points. Lysates were analysed for the endogenous levels and phosphorylation states of the specified proteins. ( d ) Torin1 shuts down mTOR activity more efficiently than starvation. MEFs were incubated in starvation media or media containing 1 μM Torin1 for the indicated amount of time. ( e ) Starvation increases phosphatase activity for ULK1 S637 in MEFs. Upper panel shows a schematic representation of the in vitro phosphatase assay. Middle panel shows outcome of the phosphatase assay comparing lysates from starved, fed or rapamycin-treated cells. Reactions were terminated at the indicated time and analysed by western blotting for ULK1 phosphorylation status. S-tag and total S6K are loading controls for the amount of substrate and enzyme, respectively, in each reaction. Lower panel shows quantitation of ULK1 S637 phosphorylation relative to time 0 from four independent experiments (fold change±s.d., n =4. two-tail Student's t -test, * P <0.05).

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: Stable Transfection, Expressing, Incubation, De-Phosphorylation Assay, Inhibition, Activity Assay, In Vitro, Phosphatase Assay, Western Blot, Quantitation Assay

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) Okadaic acid (OA) inhibits the dephosphorylation of ULK1 at S637 but not S757. MEF, U2OS or HT1080 cells were incubated in starvation media or starvation media with 200 nM OA for 1 h. Phosphorylation of S637 and S757 on ULK1 was monitored using site specific phospho-antibodies. ( b ) MEFs were treated as in a and lysates were probed for three reported mTOR substrates (S6K, 4EBP1 and DAP1) and endogenous LC3. ( c ) OA inhibits starvation-induced autophagy. MEFs stably expressing GFP-LC3, GFP-ULK1 or GFP-ATG16 were incubated in starvation media or starvation media containing 200 nM OA for 1 h. Cells were fixed and imaged for GFP-LC3, GFP-ULK1 or GFP-ATG16 translocation. Representative images from two independent experiments shown. Average number of puncta per cell were quantitated and expressed as such or as fold change relative to full media (fold change or mean±s.d., n =20. two-tail Student's t -test, * P <0.05).

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: De-Phosphorylation Assay, Incubation, Stable Transfection, Expressing, Translocation Assay

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) PP2A is required for dephosphorylation of ULK1 at S637. MEFs were transduced with lentivirus containing control shRNA or shRNA targeting the catalytic subunit of PP2A (PP2AC). 48–72 h post transduction, cells were incubated in starvation media for the indicated time and lysed for immunoblotting. ( b ) PP1C is not required for ULK1 dephosphorylation. MEF cells were treated as in a with shRNA targeting the catalytic subunit of PP1 (PP1C). ( c ) PP2AC directly dephosphorylates ULK1 S637 in vitro . Catalytic subunit of PP1 (PP1C) was included as a control. Flag-S–ULK1 substrate was incubated with 50 nM of recombinant PP2AC or PP1C at 30 °C for 20 min. ( d ) Diagram representing the PP2A Ser/Thr phosphatase complex. Regulatory subunits confer substrate specificity to the core enzyme.

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: De-Phosphorylation Assay, Transduction, shRNA, Incubation, Western Blot, In Vitro, Recombinant

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) Set up of in vitro phosphatase assay. The substrate (Flag-S–ULK1) is stable in the reaction over time and dephosphorylated only in the presence of an enzyme source (total or fractionated cell extract). ( b ) Purification scheme for the identification of the PP2A regulatory subunit from starved ULK1/2 DKO cell lysate. ( c ) Example of phosphatase assay reaction during PP2A regulatory subunit purification. Fractions from Phenyl Sepharose column were assessed for activity against ULK1. Active fractions (15–18) were combined as input for the next purification step. ( d ) Silver staining (top) and phosphatase activity assay (bottom) of fractions from the final purification step. Active fractions are boxed in red. Indicated bands were excised for mass spectrometric analysis. ( e ) Western blot showing distribution of PP2AC, B55α and ULK1 S637 phosphatase activity after a Q-column.

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: In Vitro, Phosphatase Assay, Purification, Activity Assay, Silver Staining, Western Blot

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) B55α is required for ULK1 S637 dephosphorylation. HT1080 cells stably expressing tet-inducible control shRNA, shRNA targeting the 3′UTR region of B55α (shRNA1) or shRNA targeting the coding region of B55α (shRNA2) were generated. Cells were cultured in 100 ng ml −1 Doxycycline and incubated in starvation media for the indicated amount of time. ( b ) HT1080 cells harbouring control shRNA, B55α shRNA1 or B55α shRNA1 and stable over- expression of Flag-S–B55α were incubated in starvation media for the indicated time and analysed for ULK1 dephosphorylation. ( c ) Knockdown of B55α blocks starvation-induced autophagy as monitored by florescence imaging of GFP-LC3 in HT1080 cells. Representative images from two independent experiments shown, average number of puncta per cell were quantitated and expressed as fold change relative to 0 min (fold change±s.d., n =20. two-tail Student's t -test, * P <0.05). ( d ) ULK1 interacts preferentially with B55α. 293Ts were co-transfected with Flag-S–ULK1 and GFP-B55α or GFP-PR72 (regulatory subunit from a different family). Cells were lysed and incubated with S-beads to pull down ULK1. ( e ) B55α stimulates PP2A activity towards ULK1. In vitro phosphatase assay was carried out using 5 nM of recombinant PP2AC co-purified with PRL65 and increasing amounts of B55α (10 nM or 40 nM). Coomassie brilliant blue staining of recombinant protein preparations is shown on the right.

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: De-Phosphorylation Assay, Stable Transfection, Expressing, shRNA, Generated, Cell Culture, Incubation, Over Expression, Imaging, Transfection, Activity Assay, In Vitro, Phosphatase Assay, Recombinant, Purification, Staining

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) Recombinant PP2AC–Alpha4 complex is inactive. Phosphatase assay was carried out in vitro using increasing amounts of recombinant PP2AC (3–90 nM) co-purified with Alpha4. Recombinant PP2AC-PRL65 complex (20 nM) was used as a control. Coomassie brilliant blue staining of recombinant PP2AC–Alpha4 is shown below. ( b ) Starvation induces dissociation of PP2AC from Alpha4. 293T cells were starved or incubated in complete media containing 1 μM Torin1 for 2 h. Cell lysate was prepared, crosslinked with DSP and incubated with the indicated antibodies to pull down endogenous PP2A complexes for immunoblot analysis. Quantitation of co-IPed proteins relative to full media is shown on the right. (fold change±s.d., n =3. two-tail Student's t -test, * P <0.05) ( c ) Alpha4 overexpression reduces PRL65 binding to PP2AC. 293T cells were transfected with Flag-S-Alpha4. Cells were lysed, and incubated with antibodies to pull down endogenous PP2AC. Immunoblotting was carried out to monitor the amount of PRL65 interacting with PP2AC. Panel on the right shows quantitation of PRL65 relative to untransfected cells. (fold change±s.d., n =3) ( d ) Alpha4 overexpression blocks ULK1 S637 dephosphorylation. MEF cells stably overexpressing Flag-S-Alpha4 were incubated in complete or starvation media for the indicated amount of time.

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: Recombinant, Phosphatase Assay, In Vitro, Purification, Staining, Incubation, Western Blot, Quantitation Assay, Over Expression, Binding Assay, Transfection, De-Phosphorylation Assay, Stable Transfection

Journal: Nature Communications

Article Title: Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A

doi: 10.1038/ncomms9048

Figure Lengend Snippet: ( a ) Pancreatic ductal adenocarcinoma (PDAC) cell lines have high phosphatase activity. In vitro phosphatase assay was carried out using Flag-S–ULK1 as a substrate and 10 μg of total cell lysates from indicated cancer cell lines. Left panel shows immunoblot of ULK1 S637 dephosphorylation, and right panel shows quantitation (fold change ± s.d., n =3. two-tail Student's t -test, * P <0.05). ( b , c ) PDAC cell lines have high basal autophagy. In b , cell lines were kept in complete media in the presence or absence of 20 nM bafilomycin (Baf) for 90 min and lysed for immunoblotting of endogenous LC3. In c , PDAC cell line 8988T and control cell line H460 were kept in complete media with 20 μg ml −1 cycloheximide (CHX) for the indicated time. Where indicated, 10 nM Baf was added at the start of CHX treatment. ( d ) Phosphatase activity is required for basal turnover of LC3. 8988T was treated as in c . To assess phosphatase involvement, cells were pretreated with 200 nM okadaic acid in full media for 2 h and removed at the start of CHX treatment. ( e ) ULK1 complex is required for basal LC3 turnover. 8988T cells were transduced with control shRNA or shRNA targeting ULK1. Cells were treated with 20 μg ml −1 CHX for the indicated time and immunoblotted for endogenous LC3. ( f ) ULK1 complex is required for robust proliferation of 8988T cells. Equal number of 8988T or H460 cells stably expressing control shRNA or shRNA targeting ULK1 were seeded on day 0. Upper panel shows cells fixed and stained with crystal violet on day 1 and day 4 respectively. Histograms on the lower panel show quantitation of crystal violet stain over time, relative to day 0. Error bars are standard deviation from triplicates. ( g ) ULK1 complex is required for sustained anchorage-independent growth of 8988T. Cell lines in f were used in a soft agar assay. Representative images and knockdown efficiencies are shown. Histogram shows quantitation of colonies in each cell line relative to control shRNA (relative colony number±s.d., n =3. two-tail Student's t -test, * P <0.05).

Article Snippet: Rabbit anti-total ULK1 (A7481; 1:3,000) used to detect mouse ULK1 was purchased from Sigma.

Techniques: Activity Assay, In Vitro, Phosphatase Assay, Western Blot, De-Phosphorylation Assay, Quantitation Assay, Transduction, shRNA, Stable Transfection, Expressing, Staining, Standard Deviation, Soft Agar Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective

doi: 10.3389/fnmol.2018.00109

Figure Lengend Snippet: XCT 790 modulates aggrephagy and xenophagy in mammalian cells. (A) Representative Western blot of LC3 processing assay in SH-SY5Y cells treated with XCT 790 (2 h) under growth condition and normalized LC3-II levels were quantified ( n = 3). β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and post hoc Bonferroni test. Error bars, mean ± SEM. ns-non significant, ** P < 0.01, *** P < 0.001. (B) Representative microscopy images of tandem RFP-EGFP-LC3 assay in HeLa cells treated with XCT 790 for 2 h. Yellow puncta was autophagosomes and red was autolysosomes. Fold change in autophagosomes and autolysosomes by XCT 790 were quantified ( n = 50 cells and three independent experiments). Scale bar was 15 μm. Statistical analysis was performed using one-way ANOVA and post hoc Bonferroni test. Error bars, mean ± SEM. ns-non significant, ** P < 0.01, *** P < 0.001. (C) Graph indicating the cell viability read out of SH-SY5Y overexpressing EGFP-α-synuclein treated with XCT 790 in presence of pharmacological autophagy inhibitor 3-MA. Cell viability was analyzed using CellTitre Glo (Promega) assay. More RLU readout was indicative of more cell viability and vice-versa (three independent experiments). Statistical analysis was performed using one-way ANOVA and post hoc Bonferroni test. Error bars, mean ± SEM. *** P < 0.001. (D) Graph for colony forming unit (CFU)s indicating the intracellular burden of S. typhimurium treated with XCT 790 (10 μM) for 6 h. CFU represent survival of S. typhimurium within the host cells. Fold change between the untreated and XCT 790 treated samples were quantified (three independent experiments). Statistical analysis was performed using two-tailed paired t -test. Error bars, mean ± SEM. ns-non significant, ** P < 0.01, *** P < 0.001. (E) Representative microscopy images of HeLa cells infected with mCherry expressing S. typhimurium treated with XCT 790 for 6 h. Cells were immunostained for either p62 or LC3 in untreated and XCT 790 treated samples ( n = 25 cells and three independent experiments). Scale bar 10 μm. (F) Representative Western blots of MTOR substrates—P70S6K (phospho and total form) and 4EBP1 (phospho and total form)—regulation by various treatments like XCT 790, EBSS and LiCl. β-tubulin was used as a loading control. Normalized p-P70S6K levels were quantified for three independent experiments. Statistical analysis was performed using one-way ANOVA and the post hoc Bonferroni test. Error bars, mean ± SEM. ns-non significant, * P < 0.05. (G) Representative Western blots of signaling pathway proteins like AMPK (phospho and total form) and ULK1 (phospho and total form) regulation by XCT 790 and EBSS. Normalized p-AMPK, p-ULK1 (S555), p-ULK1 (757) levels were quantified for three independent experiments. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and the post hoc Bonferroni test. Error bars, mean ± SEM. ns-non significant, * P < 0.05, ** P < 0.01, *** P < 0.001.β-tubulin was used as a loading control. Concentrations of XCT 790, 3-MA and LiCl used were 5 μM, 100 nM and 10 mM.

Article Snippet: Anti-phospho 4E-BP1T37/46 antibody (2855) and total 4E-BP1 antibody (9452), Anti-phospho P70S6K T389 antibody (9239) and total P70S6K antibody (9202), Anti-phospho AMPK antibody (8359) and total AMPK antibody (8359), Anti-phospho ULK1 antibody (8359) and total ULK1 antibody (8359) and anti-rabbit IgG, HRP (7074) antibody were purchased from Cell Signaling Technology.

Techniques: Western Blot, Microscopy, Two Tailed Test, Infection, Expressing