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    Millipore e64d
    Enhanced co-localization of cleaved caspase-8 with LC3-positive punctates in cells treated with TRAIL and cathepsin inhibitors. Bax -/- Hct116 cells were treated daily with TRAIL (A) or with TRAIL and <t>E64D/pepstatin</t> A (B). Cytospins that were prepared
    E64d, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 847 article reviews
    Price from $9.99 to $1999.99
    e64d - by Bioz Stars, 2020-08
    99/100 stars
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    e64d  (Tocris)
    93
    Tocris e64d
    NM23-H1 is degraded by lysosomal cysteine proteases, cathepsins L and B (a) Western blot analysis of lysates from serum-starved breast cancer (top) and melanoma (bottom) cell lines. HMEC=human mammary epithelial cells. HEMn=human epidermal melanocytes. LP=light pigment, DP=dark pigment. (b) Lysates from detached and attached cells treated with the proteosomal inhibitor, MG132, were probed with antibodies. (c) Western blot analysis of lysates from cells treated with lysosomal inhibitors (ammonium chloride, 20mM (BT-549) or 60mM (435s, BT-549); chloroquine, 100μM) or the cysteine protease inhibitor, <t>E64d</t> (20μM) for 8h. (d) Lysates from cell lines transfected with siRNAs were blotted with antibodies 72h after the initial transfection. Mean±SEM for (a-d) are in Supplementary Figure S2 . (e,f) Recombinant active human cathepsins L and B were incubated with recombinant NM23-H1, and reactions probed with antibodies for N- and C-termini of NM23-H1. Compared to cathepsin B, smaller amounts of cathepsin L were required to efficiently cleave NM23-H1. This is likely because the recombinant cathepsin B contained both inactive as well as active forms, and had 6X lower specific activity. (f) C-terminal cleavage products were sequenced by Edman Degradation. Input (left), and NM23-H1 sequence and cleavage site (bold; right) are shown. The site was identical for cathepsins B and L.
    E64d, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    e64d - by Bioz Stars, 2020-08
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    Image Search Results


    Enhanced co-localization of cleaved caspase-8 with LC3-positive punctates in cells treated with TRAIL and cathepsin inhibitors. Bax -/- Hct116 cells were treated daily with TRAIL (A) or with TRAIL and E64D/pepstatin A (B). Cytospins that were prepared

    Journal: Autophagy

    Article Title: Autophagic degradation of active caspase-8

    doi: 10.4161/auto.6.7.13038

    Figure Lengend Snippet: Enhanced co-localization of cleaved caspase-8 with LC3-positive punctates in cells treated with TRAIL and cathepsin inhibitors. Bax -/- Hct116 cells were treated daily with TRAIL (A) or with TRAIL and E64D/pepstatin A (B). Cytospins that were prepared

    Article Snippet: Anti-β-actin Ab (A5316) from Sigma-Aldrich; Anti-SMAC Ab (2954) was from Cell Signaling; Anti-Cox IV Ab ( ) and Alexa Fluor 488 or 647-conjugated anti-rabbit or anti-mouse Ig were from Invitrogen; Recombinant TRAIL was from PeproTech; E64D from Calbiochem; Pepstatin A and DAPI were from Sigma; Z-VAD FMK and Z-IETD-FMK were from ICN.

    Techniques:

    The co-localization of cleaved caspase-8 subunit with components of the autophagic process increases in the presence of E64D and pepstatin A. (A) Accumulation of cleaved caspase-8 in Bax -/- Hct116 cells treated with TRAIL (100 ng/ml, 5 hr) in the presence

    Journal: Autophagy

    Article Title: Autophagic degradation of active caspase-8

    doi: 10.4161/auto.6.7.13038

    Figure Lengend Snippet: The co-localization of cleaved caspase-8 subunit with components of the autophagic process increases in the presence of E64D and pepstatin A. (A) Accumulation of cleaved caspase-8 in Bax -/- Hct116 cells treated with TRAIL (100 ng/ml, 5 hr) in the presence

    Article Snippet: Anti-β-actin Ab (A5316) from Sigma-Aldrich; Anti-SMAC Ab (2954) was from Cell Signaling; Anti-Cox IV Ab ( ) and Alexa Fluor 488 or 647-conjugated anti-rabbit or anti-mouse Ig were from Invitrogen; Recombinant TRAIL was from PeproTech; E64D from Calbiochem; Pepstatin A and DAPI were from Sigma; Z-VAD FMK and Z-IETD-FMK were from ICN.

    Techniques:

    The OPA1 Q285STOP mutation induces mitophagy in MEFs. (A) Representative images of the IN Cell 1000 high content imaging acquisition system. The cells from both RedMIT-GFP-LC3 and RedMIT-GFP-LC3-OPA1 Q285STOP ) resulting in a “mask” picture (cyan: autophagosomes; red: “short” mitochondria; yellow: “long” mitochondria; purple: “colocalization” between autophagosome and mitochondria signals). The white arrows in the insets indicate the colocalization events between mitochondrial and autophagosome signal. (B) Lysosomal inhibitors E64d/Pepstatin A were added to cells growing in glucose media to block the processing of autophagolysosomes. As shown on the graph a greater accumulation of autophagosomes is observed in MEFs from the RedMITGFP-LC3-OPA1 Q285TOP mouse compared to the RedMIT-GFP-LC3-OPA1 +/+ mouse, indicating a greater flux of autophagy when the OPA1 mutation is present. (at least 500 cells counted, regression p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Validating the RedMIT/GFP-LC3 Mouse Model by Studying Mitophagy in Autosomal Dominant Optic Atrophy Due to the OPA1Q285STOP Mutation

    doi: 10.3389/fcell.2018.00103

    Figure Lengend Snippet: The OPA1 Q285STOP mutation induces mitophagy in MEFs. (A) Representative images of the IN Cell 1000 high content imaging acquisition system. The cells from both RedMIT-GFP-LC3 and RedMIT-GFP-LC3-OPA1 Q285STOP ) resulting in a “mask” picture (cyan: autophagosomes; red: “short” mitochondria; yellow: “long” mitochondria; purple: “colocalization” between autophagosome and mitochondria signals). The white arrows in the insets indicate the colocalization events between mitochondrial and autophagosome signal. (B) Lysosomal inhibitors E64d/Pepstatin A were added to cells growing in glucose media to block the processing of autophagolysosomes. As shown on the graph a greater accumulation of autophagosomes is observed in MEFs from the RedMITGFP-LC3-OPA1 Q285TOP mouse compared to the RedMIT-GFP-LC3-OPA1 +/+ mouse, indicating a greater flux of autophagy when the OPA1 mutation is present. (at least 500 cells counted, regression p

    Article Snippet: DMEM high (4.5g/L) glucose (D6546), Galactose (G5388), and the pharmacological agents E64d (E8640), Pepstatin A (77170), and Chloroquine (C6628) were purchased from Sigma Aldrich.

    Techniques: Mutagenesis, Imaging, Blocking Assay

    SHR8443 causes cell cycle arrest, autophagy, and apoptosis (A) Cell-cycle phase histograms of MCF7, MDA-MB-468, COLO205 and A549 cell lines following treatment with SHR8443 or BEZ235 at the indicated concentration for 24 h. (B) MCF7, MDA-MB-468 and A549 cells were treated with SHR8443 or BEZ235 at the indicated concentrations for 72 h, and then analyzed by annexin V-FITC/PI staining and flow cytometry. (C) After treatment of cells with SHR8443 or BEZ235 for 72 h, whole-cell lysates were immunoblotted with an anti-PARP antibody. (D) A549 cells were treated with SHR8443 (left), BEZ235, or the combination of SHR8443/BEZ235 (100 nM) with E64d/pep (10 mg/mL) for 48 h. Whole-cell lysates were analyzed by immunoblotting with an anti-LC3 antibody.

    Journal: Oncotarget

    Article Title: Pharmacologic characterization of SHR8443, a novel dual inhibitor of phosphatidylinositol 3-kinase and mammalian target of rapamycin

    doi: 10.18632/oncotarget.22439

    Figure Lengend Snippet: SHR8443 causes cell cycle arrest, autophagy, and apoptosis (A) Cell-cycle phase histograms of MCF7, MDA-MB-468, COLO205 and A549 cell lines following treatment with SHR8443 or BEZ235 at the indicated concentration for 24 h. (B) MCF7, MDA-MB-468 and A549 cells were treated with SHR8443 or BEZ235 at the indicated concentrations for 72 h, and then analyzed by annexin V-FITC/PI staining and flow cytometry. (C) After treatment of cells with SHR8443 or BEZ235 for 72 h, whole-cell lysates were immunoblotted with an anti-PARP antibody. (D) A549 cells were treated with SHR8443 (left), BEZ235, or the combination of SHR8443/BEZ235 (100 nM) with E64d/pep (10 mg/mL) for 48 h. Whole-cell lysates were analyzed by immunoblotting with an anti-LC3 antibody.

    Article Snippet: E64d/pep was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Multiple Displacement Amplification, Concentration Assay, Staining, Flow Cytometry, Cytometry

    Effect of cysteine protease inhibition with E64d on the steady state levels of BCL2-family proteins in both uninfected (MOI = 0) and infected cells (MOI 2–10) . (A) Representative immunoblots detecting the levels of the indicated BCL2-family members in response to increasing infection. Levels of calnexin are used as a loading control. (B) Quantification of the levels of specific BCL2-proteins relative to calnexin in response to increasing infecting dose. Profiles for no-treatment (NT, untreated) and treated (E64) samples are indicated along with the MOI in parentheses. The mean relative level is indicated by the symbol with the error bards representing the SE of the mean. A single asterisk above an error bar signifies a significant data point relative to the uninfected control with a p -value

    Journal: Frontiers in Microbiology

    Article Title: The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities

    doi: 10.3389/fmicb.2011.00001

    Figure Lengend Snippet: Effect of cysteine protease inhibition with E64d on the steady state levels of BCL2-family proteins in both uninfected (MOI = 0) and infected cells (MOI 2–10) . (A) Representative immunoblots detecting the levels of the indicated BCL2-family members in response to increasing infection. Levels of calnexin are used as a loading control. (B) Quantification of the levels of specific BCL2-proteins relative to calnexin in response to increasing infecting dose. Profiles for no-treatment (NT, untreated) and treated (E64) samples are indicated along with the MOI in parentheses. The mean relative level is indicated by the symbol with the error bards representing the SE of the mean. A single asterisk above an error bar signifies a significant data point relative to the uninfected control with a p -value

    Article Snippet: The E64d stock (Sigma Aldrich, Milwaukee, WI, USA, E8640) was dissolved in 50% methanol and diluted into the medium to a concentration of 100 μM.

    Techniques: Inhibition, Infection, Western Blot

    Defective autophagy and α-synuclein ( α -syn) accumulation in T lymphocytes. ( a ) Western blot analysis of LC3-II and p62 levels in T lymphocytes from patients with SLE cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus E64d and pepstatin A (Pep A). Blots shown are representative of 10 independent experiments. Densitometry analysis of LC3-II levels ( b ) and of p62 levels ( c ) relative to β -actin. Values are expressed as means±S.D. ( d ) Western blot analysis of α -syn expression in SLE T lymphocytes cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus E64d and pepstatin A. Blots shown are representative of 10 independent experiments. The bands of α -syn at 14 kDa (monomeric form) and at 70 kDa (aggregate form) are indicated by the arrows. Densitometry analyses of the monomeric form of α -syn at 14 kDa ( e ) and of the aggregate form of α -syn at 70 kDa ( f ) are shown. Values are expressed as means±S.D. * P

    Journal: Cell Death & Disease

    Article Title: Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes

    doi: 10.1038/cddis.2014.211

    Figure Lengend Snippet: Defective autophagy and α-synuclein ( α -syn) accumulation in T lymphocytes. ( a ) Western blot analysis of LC3-II and p62 levels in T lymphocytes from patients with SLE cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus E64d and pepstatin A (Pep A). Blots shown are representative of 10 independent experiments. Densitometry analysis of LC3-II levels ( b ) and of p62 levels ( c ) relative to β -actin. Values are expressed as means±S.D. ( d ) Western blot analysis of α -syn expression in SLE T lymphocytes cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus E64d and pepstatin A. Blots shown are representative of 10 independent experiments. The bands of α -syn at 14 kDa (monomeric form) and at 70 kDa (aggregate form) are indicated by the arrows. Densitometry analyses of the monomeric form of α -syn at 14 kDa ( e ) and of the aggregate form of α -syn at 70 kDa ( f ) are shown. Values are expressed as means±S.D. * P

    Article Snippet: Where indicated, cells were treated in the presence of lysosomal inhibitors E64d and pepstatin A (both at 10 μ g/ml; Sigma-Aldrich, St. Louis, MO, USA) for 2 h before the end of culture.

    Techniques: Western Blot, Cell Culture, Expressing

    Alpha-synuclein ( α -syn) degradation by autophagy in T lymphocytes. ( a ) Western blot analysis of α -syn expression in T lymphocytes cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus lysosomal inhibitors E64d and pepstatin A (Pep A). Blots shown are representative of five independent experiments. The bands of α -syn at 14 kDa (monomeric form) and at 70 kDa (aggregate form) are indicated by the arrows. Densitometry analysis of ( b ) the monomeric form of α -syn at 14 kDa ( α -syn 14 kDa/ β -actin) and of ( c ) the aggregate form of α -syn at 70 kDa ( α -syn 70 kDa/ β -actin) are shown. Values are expressed as means±S.D. * P

    Journal: Cell Death & Disease

    Article Title: Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes

    doi: 10.1038/cddis.2014.211

    Figure Lengend Snippet: Alpha-synuclein ( α -syn) degradation by autophagy in T lymphocytes. ( a ) Western blot analysis of α -syn expression in T lymphocytes cultured with (i) 10% FBS, (ii) 1% FBS and (iii) 1% FBS plus lysosomal inhibitors E64d and pepstatin A (Pep A). Blots shown are representative of five independent experiments. The bands of α -syn at 14 kDa (monomeric form) and at 70 kDa (aggregate form) are indicated by the arrows. Densitometry analysis of ( b ) the monomeric form of α -syn at 14 kDa ( α -syn 14 kDa/ β -actin) and of ( c ) the aggregate form of α -syn at 70 kDa ( α -syn 70 kDa/ β -actin) are shown. Values are expressed as means±S.D. * P

    Article Snippet: Where indicated, cells were treated in the presence of lysosomal inhibitors E64d and pepstatin A (both at 10 μ g/ml; Sigma-Aldrich, St. Louis, MO, USA) for 2 h before the end of culture.

    Techniques: Western Blot, Expressing, Cell Culture

    Morphological analysis of cardiac myocyte size in the LV wall of rats. A: Light micrographs of myocytes in H E-stained section of the LV wall of rats in the control, HF, olmesartan (OLM), and E64d groups at 19 weeks of age. B: Cross-sectional

    Journal:

    Article Title: Superoxide-Dependent Cathepsin Activation Is Associated with Hypertensive Myocardial Remodeling and Represents a Target for Angiotensin II Type 1 Receptor Blocker Treatment

    doi: 10.2353/ajpath.2008.071126

    Figure Lengend Snippet: Morphological analysis of cardiac myocyte size in the LV wall of rats. A: Light micrographs of myocytes in H E-stained section of the LV wall of rats in the control, HF, olmesartan (OLM), and E64d groups at 19 weeks of age. B: Cross-sectional

    Article Snippet: DS rats fed an 8% NaCl diet after 7 weeks manifest compensated concentric left ventricular (LV) hypertrophy secondary to hypertension at 12 weeks and a distinct stage of fatal LV failure with lung congestion at 19 weeks., DS rats were therefore fed an 8% NaCl diet from 7 weeks of age and were randomized to an HF group, an E64d group (10 mg per kg of body mass per day, administered intraperitoneally every other day; Sigma-Aldrich, St. Louis, MO), or an olmesartan group (3 mg/kg per day in chow; Sankyo Pharmaceutical, Tokyo, Japan) from 12 to 19 weeks of age ( n = 10 for each group).

    Techniques: Staining

    Effect of the cysteine protease inhibitor E64d on jin -1, wt T3D virus, and ISVP entry into cells. (A) U118MG cells were exposed to purified wt T3D virus and wt T3D ISVP (2*10 3 particles per cell). Lysates were made 24 hours post-infection and analyzed by SDS-PAGE and western-blotting. The reovirus σ3 proteins were detected by the anti-reovirus σ3 antibody 4F2 and an anti-Actin serum was used to detect actin as a loading control. (B) Effect of 100 µM E64d on the entry of particles compared to entry of ISVPs. Cells, treated (+) or untreated (−) with 100 µM E64d, were exposed to jin-1 (U118MG and 911 cells) or wt T3D (911 cells) virus or ISVPs (2*10 3 particles per cell). Lysates were made 24 hours post-infection. Equal amounts of protein were loaded on 10% SDS-polyacrylamide gel and detected with anti-reovirus σ3 antibody (4F2), and anti-Actin as a loading control.

    Journal: PLoS ONE

    Article Title: Isolation of Reovirus T3D Mutants Capable of Infecting Human Tumor Cells Independent of Junction Adhesion Molecule-A

    doi: 10.1371/journal.pone.0048064

    Figure Lengend Snippet: Effect of the cysteine protease inhibitor E64d on jin -1, wt T3D virus, and ISVP entry into cells. (A) U118MG cells were exposed to purified wt T3D virus and wt T3D ISVP (2*10 3 particles per cell). Lysates were made 24 hours post-infection and analyzed by SDS-PAGE and western-blotting. The reovirus σ3 proteins were detected by the anti-reovirus σ3 antibody 4F2 and an anti-Actin serum was used to detect actin as a loading control. (B) Effect of 100 µM E64d on the entry of particles compared to entry of ISVPs. Cells, treated (+) or untreated (−) with 100 µM E64d, were exposed to jin-1 (U118MG and 911 cells) or wt T3D (911 cells) virus or ISVPs (2*10 3 particles per cell). Lysates were made 24 hours post-infection. Equal amounts of protein were loaded on 10% SDS-polyacrylamide gel and detected with anti-reovirus σ3 antibody (4F2), and anti-Actin as a loading control.

    Article Snippet: E64d Inhibition E64d (Sigma Aldrich, Zwijndrecht The Netherlands) was dissolved in DMSO before use.

    Techniques: Protease Inhibitor, Purification, Infection, SDS Page, Western Blot

    Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 increase autophagic flux. HeLa cells were either nontreated (NT) or subjected to azetidine or canavanine (5 or 15 mM) treatment (A) or MG132 (0.5–5 μM) treatment (B) for 6 h. (C, D) Cells were left untreated (NT) or transiently transfected with control plasmids (Cont) or plasmids expressing wild-type HspB5 and its mutated form, HspB5R120G (C), or wild-type SOD1-EGFP and its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E–G) HeLa cells were subjected to the same treatments as described in the presence of 5 μM cathepsin inhibitors pepstatin A (PepA) and E64d. The inhibitors were added concomitantly to the treatment or just after transfection and were not removed until protein extraction. Total protein extracts were prepared 16 h after treatment and 24 h after transfection and subjected to SDS–PAGE. Immunoblots were probed with antibodies against LC3-I and -II, HspB5, EGFP, and actin (as a loading control). The histograms show LC3-II/actin ratios, which were calculated from quantifications of LC3-II and actin bands of Western blots by ImageJ ( n = 3; ratio was set at 1.0 for NT or Cont-transfected cells). Results are representative of three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

    doi: 10.1091/mbc.E15-12-0835

    Figure Lengend Snippet: Amino acid analogue or MG132 treatment and overexpression of mutated forms of HspB5 and SOD1 increase autophagic flux. HeLa cells were either nontreated (NT) or subjected to azetidine or canavanine (5 or 15 mM) treatment (A) or MG132 (0.5–5 μM) treatment (B) for 6 h. (C, D) Cells were left untreated (NT) or transiently transfected with control plasmids (Cont) or plasmids expressing wild-type HspB5 and its mutated form, HspB5R120G (C), or wild-type SOD1-EGFP and its mutated forms, SOD1G93A-EGFP and SOD1G85R-EGFP (D). (E–G) HeLa cells were subjected to the same treatments as described in the presence of 5 μM cathepsin inhibitors pepstatin A (PepA) and E64d. The inhibitors were added concomitantly to the treatment or just after transfection and were not removed until protein extraction. Total protein extracts were prepared 16 h after treatment and 24 h after transfection and subjected to SDS–PAGE. Immunoblots were probed with antibodies against LC3-I and -II, HspB5, EGFP, and actin (as a loading control). The histograms show LC3-II/actin ratios, which were calculated from quantifications of LC3-II and actin bands of Western blots by ImageJ ( n = 3; ratio was set at 1.0 for NT or Cont-transfected cells). Results are representative of three independent experiments.

    Article Snippet: Reagents and plasmids E64d, pepstatin A, l -azetidine-2-carboxylic acid, l -canavanine sulfate, Hoechst 33258, and Triton X-100 were from Sigma-Aldrich (Saint Quentin Fallavier, France).

    Techniques: Over Expression, Transfection, Expressing, Protein Extraction, SDS Page, Western Blot

    Overexpression of plant BI-1 trigged cell death and increased autophagic activity. (A) Trypan blue staining of cell death. Agrobacteria containing 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 were infiltrated into N. benthamiana . At 3 dpi, leaves were detached for trypan blue staining. (B) Representative images of MDC-stained autophagic structures from leaf tissues agroinfiltrated with 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 at 48 h postinfiltration (hpi), when no macroscopic cell death was apparent. (C) Representative images of CFP-ATG8F-labeled autophagic structures from leaf tissues agroinfiltrated with 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 at 48 hpi. The puncta in the mesophyll cells were autophagic structures. Scale bars ((B)and C): 5μm. (D) The relative autophagic activity in MDC-stained tissue. The number of MDC-stained autophagic structures per leaf section was counted and normalized to that of 35S:cLUC or 2 × 35S-Ω:cLUC control, respectively. Data are the means ± SEM of relative autophagic activity, with the mean value of control set to 1.0 (n = 10). Different letters (a to c) above bars represent significantly different groups. (E) The relative autophagic activity in CFP-ATG8F-labeled tissue. The number of CFP-ATG8F-labeled autophagic structures per leaf section was counted and normalized to control respectively. Data are the means ± SEM of relative autophagic activity, with the mean value of control set to 1.0 (n = 10). Different letters (a to c) above bars represent significantly different groups. (F) Overexpression of AtBI-1 decreased the accumulation of JOKA2. Ponceau S staining was used as loading control. Experiments were repeated at least 2 times. All autophagy-related experiments were performed with E64d treatment.

    Journal: Autophagy

    Article Title: Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death

    doi: 10.1080/15548627.2017.1320633

    Figure Lengend Snippet: Overexpression of plant BI-1 trigged cell death and increased autophagic activity. (A) Trypan blue staining of cell death. Agrobacteria containing 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 were infiltrated into N. benthamiana . At 3 dpi, leaves were detached for trypan blue staining. (B) Representative images of MDC-stained autophagic structures from leaf tissues agroinfiltrated with 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 at 48 h postinfiltration (hpi), when no macroscopic cell death was apparent. (C) Representative images of CFP-ATG8F-labeled autophagic structures from leaf tissues agroinfiltrated with 35S:cLUC , 2 × 35S-Ω:cLUC, 35S:AtBI-1 , and 2 × 35S-Ω:AtBI-1 at 48 hpi. The puncta in the mesophyll cells were autophagic structures. Scale bars ((B)and C): 5μm. (D) The relative autophagic activity in MDC-stained tissue. The number of MDC-stained autophagic structures per leaf section was counted and normalized to that of 35S:cLUC or 2 × 35S-Ω:cLUC control, respectively. Data are the means ± SEM of relative autophagic activity, with the mean value of control set to 1.0 (n = 10). Different letters (a to c) above bars represent significantly different groups. (E) The relative autophagic activity in CFP-ATG8F-labeled tissue. The number of CFP-ATG8F-labeled autophagic structures per leaf section was counted and normalized to control respectively. Data are the means ± SEM of relative autophagic activity, with the mean value of control set to 1.0 (n = 10). Different letters (a to c) above bars represent significantly different groups. (F) Overexpression of AtBI-1 decreased the accumulation of JOKA2. Ponceau S staining was used as loading control. Experiments were repeated at least 2 times. All autophagy-related experiments were performed with E64d treatment.

    Article Snippet: The MDC staining assay was performed as described; the leaves were infiltrated with 100 μM E64d (Sigma-Aldrich, E8640) and treated in the dark for 10 to 12 h. After the dark treatment, the E64d-infiltrated parts of the leaves were excised and immediately vacuum infiltrated with 50 μM MDC (Sigma-Aldrich, 30432) for 10 min, followed by 2 washes with phosphate-buffered saline buffer pH 7.4 (137 mM NaCl [Sinopharm Chemical Reagent, 10019308], 2.7 mM KCl [Sinopharm Chemical Reagent, 10016308], 10 mM Na2 HPO4 [Sinopharm Chemical Reagent, 20040618], 1.8 mM KH2 PO4 [Sinopharm Chemical Reagent, 10017608]).

    Techniques: Over Expression, Activity Assay, Staining, Labeling

    Candida albicans treatment decreases autophagy via an MTOR-independent mechanism. (a) THP-1-derived macrophages were treated with or without C. albicans and rapamycin for 8 hours in the presence or absence of E-64d and pepstatin. The level of LC3 protein was determined by Western blotting. The ratio of LC3-II/ β -actin was calculated. (b, c) THP-1-derived macrophages were treated with or without C. albicans and torin1/pp242 for 8 hours. The level of MTORC1 and MTORC2 activity was detected using Western blotting. Bars represent the mean ± SEM of n = 3 independent experiments ( ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Phagocytosis of Candida albicans Inhibits Autophagic Flux in Macrophages

    doi: 10.1155/2018/4938649

    Figure Lengend Snippet: Candida albicans treatment decreases autophagy via an MTOR-independent mechanism. (a) THP-1-derived macrophages were treated with or without C. albicans and rapamycin for 8 hours in the presence or absence of E-64d and pepstatin. The level of LC3 protein was determined by Western blotting. The ratio of LC3-II/ β -actin was calculated. (b, c) THP-1-derived macrophages were treated with or without C. albicans and torin1/pp242 for 8 hours. The level of MTORC1 and MTORC2 activity was detected using Western blotting. Bars represent the mean ± SEM of n = 3 independent experiments ( ∗ P

    Article Snippet: The compounds used in this study included E-64d (E8640), pepstatin (P5318), rapamycin (V900930), chloroquine (C6628), dimethyl sulfoxide (D2650), and acridine orange (AO, A8097) (all from Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Derivative Assay, Western Blot, Activity Assay

    Candida albicans treatment reduces autophagic flux. (a) THP-1-derived macrophages were treated with C. albicans in the presence or absence of chloroquine (CQ) for the indicated time. The LC3-II/ β -actin ratio decreased after treatment with C. albicans . A significant difference in the LC3-II/ β -actin ratio was not observed between dimethyl sulfoxide- (DMSO-) treated and untreated cells. (b, c, d) THP-1-derived macrophages were treated with C. albicans in the presence or absence of E-64d+ pepstatin, NH 4 Cl, and BAF-A1 for the indicated period. The LC3-II/ β -actin ratio decreased after treatment with C. albicans . (e, f) RAW 264.7 macrophage-like cells were treated with C. albicans in the presence or absence of NH 4 Cl/BAF-A1 for 8 hours. The LC3-II/ β -actin ratio was decreased after treatment with C. albicans. Bars represent the mean ± SEM of n = 3 independent experiments performed in triplicate ( ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Phagocytosis of Candida albicans Inhibits Autophagic Flux in Macrophages

    doi: 10.1155/2018/4938649

    Figure Lengend Snippet: Candida albicans treatment reduces autophagic flux. (a) THP-1-derived macrophages were treated with C. albicans in the presence or absence of chloroquine (CQ) for the indicated time. The LC3-II/ β -actin ratio decreased after treatment with C. albicans . A significant difference in the LC3-II/ β -actin ratio was not observed between dimethyl sulfoxide- (DMSO-) treated and untreated cells. (b, c, d) THP-1-derived macrophages were treated with C. albicans in the presence or absence of E-64d+ pepstatin, NH 4 Cl, and BAF-A1 for the indicated period. The LC3-II/ β -actin ratio decreased after treatment with C. albicans . (e, f) RAW 264.7 macrophage-like cells were treated with C. albicans in the presence or absence of NH 4 Cl/BAF-A1 for 8 hours. The LC3-II/ β -actin ratio was decreased after treatment with C. albicans. Bars represent the mean ± SEM of n = 3 independent experiments performed in triplicate ( ∗ P

    Article Snippet: The compounds used in this study included E-64d (E8640), pepstatin (P5318), rapamycin (V900930), chloroquine (C6628), dimethyl sulfoxide (D2650), and acridine orange (AO, A8097) (all from Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Derivative Assay

    Lung I/R injury induces autophagy in lung tissues of minipigs. ( a ) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. ( b ) Immunoblotting analysis of LC3, BECN1, ATG5 and β -actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. ( c ) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. ( d ) Immunoblotting analysis of LC3, BECN1, ATG7 and β -actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. ( e ) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. ( f ) Immunoblotting analysis of LC3 and β -actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μ g/ml) and Pepstatin A (15 μ g/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β -actin in the same lane. The relative band intensities of LC3-II/ β -actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments ( b – f ). * P

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced by DAMPs facilitates the inflammation response in lungs undergoing ischemia-reperfusion injury through promoting TRAF6 ubiquitination

    doi: 10.1038/cdd.2017.1

    Figure Lengend Snippet: Lung I/R injury induces autophagy in lung tissues of minipigs. ( a ) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. ( b ) Immunoblotting analysis of LC3, BECN1, ATG5 and β -actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. ( c ) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. ( d ) Immunoblotting analysis of LC3, BECN1, ATG7 and β -actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. ( e ) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. ( f ) Immunoblotting analysis of LC3 and β -actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μ g/ml) and Pepstatin A (15 μ g/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β -actin in the same lane. The relative band intensities of LC3-II/ β -actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments ( b – f ). * P

    Article Snippet: Reagents E64d (E8640), Pepstatin A (P4265), Bafilomycin A1 (B1793) and 3-MA (M9281) were from Sigma (St. Louis, MO, USA).

    Techniques: Immunohistochemistry, Staining, Immunofluorescence, Software

    DAMPs are involved in lung I/R injury-triggered autophagy in alveolar macrophages of minipigs. ( a ) ELISA of HMGB1 and HSP60 production in the BALF of the sham group or minipigs subjected to lung I/R as indicated ( n =3). ( b ) Immunohistochemical staining of HMGB1 and HSP60 in left lung tissues of the sham group or minipigs subjected to lung I/R as indicated. ( c ) Immunoblotting analysis of LC3, BECN1, SQSTM1 and β -actin (as a loading control) in lysates of alveolar macrophages treated with rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for the indicated periods. ( d ) Immunofluorescence analysis of LC3 in alveolar macrophages stimulated with rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for 3 h. Original magnification, × 630. Quantification of cells with autophagosomes is also shown. ( e ) Immunoblotting analysis of LC3, SQSTM1 and β -actin (as a loading control) in lysates of alveolar macrophages with or without treatment with E64d (15 μ g/ml), Pepstatin A (15 μ g/ml) or Bafilomycin A 1 (30 nM) before stimulation by rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for 3 h. Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1 and SQSTM1) to β -actin in the same lane. The relative band intensities of LC3-II/ β -actin were calculated from three independent experiments and shown as mean±S.E.M. Data are mean±S.E.M. ( a ) or representative ( b – e ), of three individual experiments. * P

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced by DAMPs facilitates the inflammation response in lungs undergoing ischemia-reperfusion injury through promoting TRAF6 ubiquitination

    doi: 10.1038/cdd.2017.1

    Figure Lengend Snippet: DAMPs are involved in lung I/R injury-triggered autophagy in alveolar macrophages of minipigs. ( a ) ELISA of HMGB1 and HSP60 production in the BALF of the sham group or minipigs subjected to lung I/R as indicated ( n =3). ( b ) Immunohistochemical staining of HMGB1 and HSP60 in left lung tissues of the sham group or minipigs subjected to lung I/R as indicated. ( c ) Immunoblotting analysis of LC3, BECN1, SQSTM1 and β -actin (as a loading control) in lysates of alveolar macrophages treated with rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for the indicated periods. ( d ) Immunofluorescence analysis of LC3 in alveolar macrophages stimulated with rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for 3 h. Original magnification, × 630. Quantification of cells with autophagosomes is also shown. ( e ) Immunoblotting analysis of LC3, SQSTM1 and β -actin (as a loading control) in lysates of alveolar macrophages with or without treatment with E64d (15 μ g/ml), Pepstatin A (15 μ g/ml) or Bafilomycin A 1 (30 nM) before stimulation by rpHMGB1 (1 μ g/ml) or rpHSP60 (1 μ g/ml) for 3 h. Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1 and SQSTM1) to β -actin in the same lane. The relative band intensities of LC3-II/ β -actin were calculated from three independent experiments and shown as mean±S.E.M. Data are mean±S.E.M. ( a ) or representative ( b – e ), of three individual experiments. * P

    Article Snippet: Reagents E64d (E8640), Pepstatin A (P4265), Bafilomycin A1 (B1793) and 3-MA (M9281) were from Sigma (St. Louis, MO, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Immunofluorescence

    Autophagy controls intracellular MB d levels. ( a ) Single-plane confocal images of MB d s within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MB d markers: Cep55, MKLP1, or mgcRACGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and mgcRACGAP (red) are co-localized (magenta) in the autophagosome (green), suggesting that MB d s are sorted into autophagosomes. Bars, 2 μm. ( b ) Decreasing autophagy levels by deletion of Atg5 gene (left, MEFs) or depletion of Atg7 by siRNA (right, HeLa) significantly increases the percent of MB d + cells (p=0.0019 and p=0.021, respectively, n =3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of Atg7 (asterisk). ( c ) Rapamycin (Rapa) and lithium chloride (LiCl) co-treatment induces autophagy and decreases the percent of MB d + cells (left, HeLa; p=0.0056, n =3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by over-expression of Flag-tagged BECN1 reduces the percent of MB d + cells (right, MCF-7; p=0.0008, n =4) ( d ) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d/PepA. U, uninhibited. I, inhibited. Below, the average of the percent change in LC3-II levels after lysosomal inhibition from 3 experiments. α-tubulin, loading control. ( e ) Quantification of autophagic flux from 3 experiments in different cell lines. Normal dividing cells (MB d -poor) typically have high autophagic flux, whereas stem and cancer cells (MB d -rich) have low autophagic flux. The data are presented as mean ± s.d. (b-e).

    Journal: Nature cell biology

    Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

    doi: 10.1038/ncb2332

    Figure Lengend Snippet: Autophagy controls intracellular MB d levels. ( a ) Single-plane confocal images of MB d s within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MB d markers: Cep55, MKLP1, or mgcRACGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and mgcRACGAP (red) are co-localized (magenta) in the autophagosome (green), suggesting that MB d s are sorted into autophagosomes. Bars, 2 μm. ( b ) Decreasing autophagy levels by deletion of Atg5 gene (left, MEFs) or depletion of Atg7 by siRNA (right, HeLa) significantly increases the percent of MB d + cells (p=0.0019 and p=0.021, respectively, n =3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of Atg7 (asterisk). ( c ) Rapamycin (Rapa) and lithium chloride (LiCl) co-treatment induces autophagy and decreases the percent of MB d + cells (left, HeLa; p=0.0056, n =3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by over-expression of Flag-tagged BECN1 reduces the percent of MB d + cells (right, MCF-7; p=0.0008, n =4) ( d ) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d/PepA. U, uninhibited. I, inhibited. Below, the average of the percent change in LC3-II levels after lysosomal inhibition from 3 experiments. α-tubulin, loading control. ( e ) Quantification of autophagic flux from 3 experiments in different cell lines. Normal dividing cells (MB d -poor) typically have high autophagic flux, whereas stem and cancer cells (MB d -rich) have low autophagic flux. The data are presented as mean ± s.d. (b-e).

    Article Snippet: Cells at 70% confluency were incubated with chloroquine (200 μM/PBS; Sigma), E64d + pepstatin A (E64d/PepA) (10 μg ml-1 /DMSO each; Sigma) , or solvents alone (controls) for 22 hours before fixation.

    Techniques: Expressing, Staining, Western Blot, Conjugation Assay, Mutagenesis, Over Expression, Activity Assay, Inhibition

    WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL Pepstatin A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL Pepstatin A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Incubation, Plaque Assay, Expressing

    LC3B-II accumulation in 293T cells. (A) Effect of WNV infection on LC3-II levels. Whole cell lysates were prepared from mock-infected or WNV-infected (MOI = 3) 293T cells at 24 hours post-infection. Immunoblot anyalysis was used to determine steady-state levels of LC3B (top), GAPDH (middle), and WNV (bottom). (B) Effect of rapamycin on LC3B. Whole cell lysates prepared at the indicated times (h) from 293T cells treated with 100 nM rapamycin (rap) were subjected to immunoblot analysis to determine the steady-state levels of LC3B (top) and GAPDH (bottom). (C) Effect of protease inhibitors on LC3B-II levels. Whole cell lysates were prepared from 293T monolayers treated with rapamycin (Rap) in the presence or absence of pepstatin A (Pep) and/or E64d at 24 hours post-treatment. Lysates were subjected to immunoblot analysis as described in Panel B.

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: LC3B-II accumulation in 293T cells. (A) Effect of WNV infection on LC3-II levels. Whole cell lysates were prepared from mock-infected or WNV-infected (MOI = 3) 293T cells at 24 hours post-infection. Immunoblot anyalysis was used to determine steady-state levels of LC3B (top), GAPDH (middle), and WNV (bottom). (B) Effect of rapamycin on LC3B. Whole cell lysates prepared at the indicated times (h) from 293T cells treated with 100 nM rapamycin (rap) were subjected to immunoblot analysis to determine the steady-state levels of LC3B (top) and GAPDH (bottom). (C) Effect of protease inhibitors on LC3B-II levels. Whole cell lysates were prepared from 293T monolayers treated with rapamycin (Rap) in the presence or absence of pepstatin A (Pep) and/or E64d at 24 hours post-treatment. Lysates were subjected to immunoblot analysis as described in Panel B.

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection

    The autophagy pathway is not upregulated by WNV in established cell lines. (A and B) Effect of WNV infection on steady-state LC3B-II levels in Huh7 (A) or Huh7.5 (B) cells. Following mock or WNV-NY (MOI = 3) infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV and SINV infection of steady-state LC3B-II and p62 levels in Neuro2A cells. Neuro2A cells were mock-infected or infected with WNV (MOI = 3) or SINV (MOI = 5) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), WNV (third panel), and SINV (bottom panel).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: The autophagy pathway is not upregulated by WNV in established cell lines. (A and B) Effect of WNV infection on steady-state LC3B-II levels in Huh7 (A) or Huh7.5 (B) cells. Following mock or WNV-NY (MOI = 3) infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV and SINV infection of steady-state LC3B-II and p62 levels in Neuro2A cells. Neuro2A cells were mock-infected or infected with WNV (MOI = 3) or SINV (MOI = 5) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), WNV (third panel), and SINV (bottom panel).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Expressing

    Atg5 is not required for WNV replication. (A) Atg5 mRNA in m5-7 cells. Standard RT-PCR (+ RT) was used to detect the presence of atg5 (top) or gapdh (bottom) mRNA in total RNA extracted from m5-7 cells grown in the presence or absence of 10 ng/mL doxycycline (Dox). PCR products were analyzed by agarose-gel electrophoresis. (B) Atg5 protein expression in m5-7 cells. Whole cell lysates prepared from m5-7 cells grown in the presence or absence of doxycycline were subjected to immunoblot analysis for Atg5 expression. A non-specific band (*) was also detected with this antiserum. (C) Immunoblot analysis of WNV-NY-infected m5-7 cells. m5-7 monolayers grown in the presence (Atg5 - ) or absence (Atg5 + ) of 10 ng/mL doxycycline were mock-infected or infected with WNV-NY (MOI = 3). After infection, inoculum was replaced with medium containing 10 µg/mL Peptastatin and E64d with or without 10 ng/mL doxycycline. Whole cell lysates were prepared at the indicated times (h) post-infection and subjected to immunoblot analysis for expression of LC3 (top), WNV (middle) and GAPDH (bottom). (D) WNV growth curves in the presence and absence of Atg5. Culture supernatants were recovered from the cells in Panel C at the indicated times (h) and the viral titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from four independent experiments.

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: Atg5 is not required for WNV replication. (A) Atg5 mRNA in m5-7 cells. Standard RT-PCR (+ RT) was used to detect the presence of atg5 (top) or gapdh (bottom) mRNA in total RNA extracted from m5-7 cells grown in the presence or absence of 10 ng/mL doxycycline (Dox). PCR products were analyzed by agarose-gel electrophoresis. (B) Atg5 protein expression in m5-7 cells. Whole cell lysates prepared from m5-7 cells grown in the presence or absence of doxycycline were subjected to immunoblot analysis for Atg5 expression. A non-specific band (*) was also detected with this antiserum. (C) Immunoblot analysis of WNV-NY-infected m5-7 cells. m5-7 monolayers grown in the presence (Atg5 - ) or absence (Atg5 + ) of 10 ng/mL doxycycline were mock-infected or infected with WNV-NY (MOI = 3). After infection, inoculum was replaced with medium containing 10 µg/mL Peptastatin and E64d with or without 10 ng/mL doxycycline. Whole cell lysates were prepared at the indicated times (h) post-infection and subjected to immunoblot analysis for expression of LC3 (top), WNV (middle) and GAPDH (bottom). (D) WNV growth curves in the presence and absence of Atg5. Culture supernatants were recovered from the cells in Panel C at the indicated times (h) and the viral titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from four independent experiments.

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Infection, Plaque Assay

    WNV-MAD78 infection does not induce autophagy. 293T monolayers were mock-infected or infected with WNV-MAD78 (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for LC3B (top), GAPDH (middle) and WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: WNV-MAD78 infection does not induce autophagy. 293T monolayers were mock-infected or infected with WNV-MAD78 (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for LC3B (top), GAPDH (middle) and WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection

    Primary human cell lines do not upregulate autophagy in response to WNV infection. (A-B) Effect of WNV infection on steady-state LC3B-II levels. Human foreskin fibroblasts (A) or human brain cortical astrocytes (B) were mock-infected or infected with WNV-NY (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle) and WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: Primary human cell lines do not upregulate autophagy in response to WNV infection. (A-B) Effect of WNV infection on steady-state LC3B-II levels. Human foreskin fibroblasts (A) or human brain cortical astrocytes (B) were mock-infected or infected with WNV-NY (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle) and WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Expressing

    Inhibition of autophagy increases pemetrexed and simvastatin-induced apoptosis Cells were treated with 1 μM pemetrexed and 5 μM simvastatin in the absence or presence of the autophagy inhibitors 1 mM 3-MA A. ATG5 siRNA C. 50 nM bafilomycin A E. and 10 μM E64d/pepstatin A G. The cell lysates were subjected to 12% SDS-PAGE to measure the expression of indicated proteins. B, D, F. and H. Apoptosis was evaluated as described in Figure 2A . The data represent the mean ± SD of three independent experiments. * p

    Journal: Oncotarget

    Article Title: Inhibition of autophagy potentiates pemetrexed and simvastatin-induced apoptotic cell death in malignant mesothelioma and non-small cell lung cancer cells

    doi:

    Figure Lengend Snippet: Inhibition of autophagy increases pemetrexed and simvastatin-induced apoptosis Cells were treated with 1 μM pemetrexed and 5 μM simvastatin in the absence or presence of the autophagy inhibitors 1 mM 3-MA A. ATG5 siRNA C. 50 nM bafilomycin A E. and 10 μM E64d/pepstatin A G. The cell lysates were subjected to 12% SDS-PAGE to measure the expression of indicated proteins. B, D, F. and H. Apoptosis was evaluated as described in Figure 2A . The data represent the mean ± SD of three independent experiments. * p

    Article Snippet: 3-Methyl adenine (3-MA), bafilomycin A, and E64D/pepstatin A were purchased from Sigma-Aldrich.

    Techniques: Inhibition, SDS Page, Expressing

    Effect of cathepsin inhibitors on the processing, activity and endocytic trafficking in T47D-MPO cells. (A) T47D-MPO cells were grown with or without 8 μM ALLN for 44 h. MPO was partially purified from the resulting cell extracts on SP-sepharose and analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (left panel) or antibodies specific for epitopes within the light chain of MPO (right panel). (B) T47D-MPO cells were grown in the presence of the indicated concentrations of e64d for 44 h and the resulting cell extracts analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody. (C) Samples analyzed in panel A were assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Methods and Materials”. (D) T47D-MPO cells were grown on glass coverslips in the presence of 8 μM ALLN for 72 h. The treated cells were fixed, permeabolized and double-labeled with antibodies against MPO (red) and Lamp1 (green). Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope.

    Journal: PLoS ONE

    Article Title: T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    doi: 10.1371/journal.pone.0149391

    Figure Lengend Snippet: Effect of cathepsin inhibitors on the processing, activity and endocytic trafficking in T47D-MPO cells. (A) T47D-MPO cells were grown with or without 8 μM ALLN for 44 h. MPO was partially purified from the resulting cell extracts on SP-sepharose and analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (left panel) or antibodies specific for epitopes within the light chain of MPO (right panel). (B) T47D-MPO cells were grown in the presence of the indicated concentrations of e64d for 44 h and the resulting cell extracts analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody. (C) Samples analyzed in panel A were assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Methods and Materials”. (D) T47D-MPO cells were grown on glass coverslips in the presence of 8 μM ALLN for 72 h. The treated cells were fixed, permeabolized and double-labeled with antibodies against MPO (red) and Lamp1 (green). Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope.

    Article Snippet: The cathepsin inhibitors e64d (E8640) and N-acetyl-Leucine-Leucine-Norleucine (208719) were obtained from Sigma and Calbiochem respectively.

    Techniques: Activity Assay, Purification, Labeling, Microscopy

    Representative images of mossy fiber sprouting by Timm staining in the (A) CONT, (B) RS and (C) E64D groups. Parts labeled as (a) represent panoramic view of hippocampus from CONT to E4D; (b) represent CA3 subfield from CONT to E64D; similarly, parts labeled as (c) represent dentate gyrus subfield. Excessive quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in B-b) and in the inner molecular layer of the granule cells (as shown in B-b) in the RS group (arrows). Mild quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in C-b) in the E64D group (arrow). Data are expressed as the mean ± standard deviation. (D) Timm staining scores compared using a non-parametric Kruskal-Wallis test. Magnification: (a) ×40; (b) and (c) ×100. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Long-term expression of zinc transporters in hippocampus following penicillin-induced developmental seizures and its regulation by E-64d

    doi: 10.3892/etm.2016.3276

    Figure Lengend Snippet: Representative images of mossy fiber sprouting by Timm staining in the (A) CONT, (B) RS and (C) E64D groups. Parts labeled as (a) represent panoramic view of hippocampus from CONT to E4D; (b) represent CA3 subfield from CONT to E64D; similarly, parts labeled as (c) represent dentate gyrus subfield. Excessive quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in B-b) and in the inner molecular layer of the granule cells (as shown in B-b) in the RS group (arrows). Mild quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in C-b) in the E64D group (arrow). Data are expressed as the mean ± standard deviation. (D) Timm staining scores compared using a non-parametric Kruskal-Wallis test. Magnification: (a) ×40; (b) and (c) ×100. *P

    Article Snippet: E64D group rats were pretreated with E-64d (Sigma-Aldrich, St. Louis, MO, USA) prior to seizure induction (4 µl, 1 mg/µl/day, i.p.).

    Techniques: Staining, Labeling, Standard Deviation

    PPRV infection enhances autophagic flux. (a) EECs were mock-infected or infected with PPRV (MOI = 1) for 24, 48, 72 and 96 h. The cell samples were then analysed by immunoblotting with anti-p62 and anti-β-actin (loading control) antibodies. (b) The relative quantification of p62 protein levels compared to β-actin protein levels was determined by densitometry in PPRV-infected cells. (c and d) Cells mock-infected with PBS, cells pre-treated with rapamycin for 12 h and then mock-infected with PBS, and cells infected with PPRV (MOI = 1) were further cultured in the absence and presence of 10 mg/mL E64d for 48 and 72 h. The cell samples were then analysed by immunoblotting with anti-PPRV-N, anti-LC3, anti-p62, and anti-β-actin (loading control) antibodies. (e and f) The relative quantification of LC3 and p62 protein levels compared to β-actin protein levels was determined by densitometry in mock-infected, rapamycin-pre-treated, and PPRV-infected EECs in the absence and presence of E64d. (g) The relative quantification of N protein levels compared to β-actin protein levels was determined by densitometry in PPRV-infected EECs in the absence and presence of E64d. (h) EECs were mock-infected or infected with PPRV (MOI = 1) for 72 h. Then, the cells were fixed and processed for indirect immunofluorescence using antibodies against LC3 and LAMP1 protein. The cell nuclei were counterstained with Hoechst 33342. 3D surface rendered (SR) images in the boxed area are shown in the right panels. Scale bars, 20 μm. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; * P

    Journal: Virulence

    Article Title: Autophagy enhances the replication of Peste des petits ruminants virus and inhibits caspase-dependent apoptosis in vitro

    doi: 10.1080/21505594.2018.1496776

    Figure Lengend Snippet: PPRV infection enhances autophagic flux. (a) EECs were mock-infected or infected with PPRV (MOI = 1) for 24, 48, 72 and 96 h. The cell samples were then analysed by immunoblotting with anti-p62 and anti-β-actin (loading control) antibodies. (b) The relative quantification of p62 protein levels compared to β-actin protein levels was determined by densitometry in PPRV-infected cells. (c and d) Cells mock-infected with PBS, cells pre-treated with rapamycin for 12 h and then mock-infected with PBS, and cells infected with PPRV (MOI = 1) were further cultured in the absence and presence of 10 mg/mL E64d for 48 and 72 h. The cell samples were then analysed by immunoblotting with anti-PPRV-N, anti-LC3, anti-p62, and anti-β-actin (loading control) antibodies. (e and f) The relative quantification of LC3 and p62 protein levels compared to β-actin protein levels was determined by densitometry in mock-infected, rapamycin-pre-treated, and PPRV-infected EECs in the absence and presence of E64d. (g) The relative quantification of N protein levels compared to β-actin protein levels was determined by densitometry in PPRV-infected EECs in the absence and presence of E64d. (h) EECs were mock-infected or infected with PPRV (MOI = 1) for 72 h. Then, the cells were fixed and processed for indirect immunofluorescence using antibodies against LC3 and LAMP1 protein. The cell nuclei were counterstained with Hoechst 33342. 3D surface rendered (SR) images in the boxed area are shown in the right panels. Scale bars, 20 μm. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; * P

    Article Snippet: Additionally, rapamycin (R0395), NH4 Cl (V900222), chloroquine (C6628), protease inhibitor E64d (E8640) and Hoechst 33342 (B2261) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Infection, Cell Culture, Immunofluorescence

    NM23-H1 is degraded by lysosomal cysteine proteases, cathepsins L and B (a) Western blot analysis of lysates from serum-starved breast cancer (top) and melanoma (bottom) cell lines. HMEC=human mammary epithelial cells. HEMn=human epidermal melanocytes. LP=light pigment, DP=dark pigment. (b) Lysates from detached and attached cells treated with the proteosomal inhibitor, MG132, were probed with antibodies. (c) Western blot analysis of lysates from cells treated with lysosomal inhibitors (ammonium chloride, 20mM (BT-549) or 60mM (435s, BT-549); chloroquine, 100μM) or the cysteine protease inhibitor, E64d (20μM) for 8h. (d) Lysates from cell lines transfected with siRNAs were blotted with antibodies 72h after the initial transfection. Mean±SEM for (a-d) are in Supplementary Figure S2 . (e,f) Recombinant active human cathepsins L and B were incubated with recombinant NM23-H1, and reactions probed with antibodies for N- and C-termini of NM23-H1. Compared to cathepsin B, smaller amounts of cathepsin L were required to efficiently cleave NM23-H1. This is likely because the recombinant cathepsin B contained both inactive as well as active forms, and had 6X lower specific activity. (f) C-terminal cleavage products were sequenced by Edman Degradation. Input (left), and NM23-H1 sequence and cleavage site (bold; right) are shown. The site was identical for cathepsins B and L.

    Journal: Oncogene

    Article Title: c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer

    doi: 10.1038/onc.2013.399

    Figure Lengend Snippet: NM23-H1 is degraded by lysosomal cysteine proteases, cathepsins L and B (a) Western blot analysis of lysates from serum-starved breast cancer (top) and melanoma (bottom) cell lines. HMEC=human mammary epithelial cells. HEMn=human epidermal melanocytes. LP=light pigment, DP=dark pigment. (b) Lysates from detached and attached cells treated with the proteosomal inhibitor, MG132, were probed with antibodies. (c) Western blot analysis of lysates from cells treated with lysosomal inhibitors (ammonium chloride, 20mM (BT-549) or 60mM (435s, BT-549); chloroquine, 100μM) or the cysteine protease inhibitor, E64d (20μM) for 8h. (d) Lysates from cell lines transfected with siRNAs were blotted with antibodies 72h after the initial transfection. Mean±SEM for (a-d) are in Supplementary Figure S2 . (e,f) Recombinant active human cathepsins L and B were incubated with recombinant NM23-H1, and reactions probed with antibodies for N- and C-termini of NM23-H1. Compared to cathepsin B, smaller amounts of cathepsin L were required to efficiently cleave NM23-H1. This is likely because the recombinant cathepsin B contained both inactive as well as active forms, and had 6X lower specific activity. (f) C-terminal cleavage products were sequenced by Edman Degradation. Input (left), and NM23-H1 sequence and cleavage site (bold; right) are shown. The site was identical for cathepsins B and L.

    Article Snippet: Imatinib and nilotinib were provided by Novartis (Basel, Switzerland); IGF-1 was from Upstate Biotechnology (Charlottesville, VA), matrigel invasion chambers and collagen I were from BD Biosciences; chloroquine and ammonium chloride were from Sigma; MG132 and proteasome inhibitor I (PS1) were from Millipore; and E64d was from Tocris (Minneapolis, MN).

    Techniques: Western Blot, Protease Inhibitor, Transfection, Recombinant, Incubation, Activity Assay, Sequencing