Journal: Journal of Biological Chemistry

Article Title: Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling

doi: 10.1074/jbc.m114.608471

Figure Lengend Snippet: FIGURE 2. ReducedenduranceandalteredautophagicsignalinginskeletalmuscleofTpcn2/mice.A,mice(male;4–5monthsold)weretestedonatreadmill underdietaryconditionsofregularchowor3-dayfasting.Thedistancestraveled(inmeters)untilexhaustionwererecordedandanalyzed(n6mice/group).Dataare means S.E. (*, p 0.05; **, p 0.01). B, autophagy flux studies under regular diet or extended starvation. Mice (male; 4–5 months old) were fed with regular chow or fasted for 3 days. The TA muscles were then harvested and homogenized for immunoblotting with p-mTOR, total mTOR (t-mTOR), p-AKT, total AKT (t-AKT), p62, LAMP1, and -tubulin. C–F, densitometry quantification of mTOR activity (p-mTOR/total mTOR; C), AKT activity (p-AKT/total AKT; D), normalized (over -tubulin) p62 (E), and LAMP1 (F). Error bars represent S.E. (n 6 mice/group; *, p 0.05; **, p 0.01; NS, not significant; analysis of variance).

Article Snippet: Antibodies for Western blotting were as follows: -tubulin (Sigma), LC3 (clone 5F10, Nanotools), lysosome-associated membrane protein-1 (LAMP1) (Santa Cruz Biotechnology), HA (clone12CA5, Roche Applied Science), dystrophin (Spring Bioscience), p62/ sequestosome-1 (SQSTM1) (Novus Biological), S6K, phosphoS6K (phospho-Thr-389), S6RP (ribosomal protein), phosphoS6RP (phospho-Ser-235/236), AKT and phospho-AKT, and mTOR and phospho-mTOR (phospho-Ser-2448) (all from Cell Signaling Technology).

Techniques: Muscles, Western Blot, Activity Assay

Journal: Cell Death & Disease

Article Title: Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma

doi: 10.1038/cddis.2017.187

Figure Lengend Snippet: Fragmented mtDNA induces macrophage autophagy. ( a ) Western blot of whole-cell lysates showing LC3 expression in BMDM stimulated by 40 μ l/ml BCM for 0–24 h. GAPDH used as loading control. ( b ) Western blot of whole-cell lysates showing LC3 expression in BMDM treated with 40 μ l/ml BCM or 10 μ g/ml CIRP for 24 h +/− IgG isotype antibody (10 μ g/ml) or CIRP neutralizing antibody (10 μ g/ml). ( c ) Western blot of whole-cell lysates showing LC3 expession in WT or gp91 phox −/− BMDM stimulated by 40 μ l/ml BCM or 10 μ g/ml CIRP for 24 h. ( d ) Western blot of whole-cell lysates showing LC3 expression in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μ l/ml) for 24 h. ( e ) Confocal immunofluorescence images and analysis showing colocalization of LC3 (autophagosome; green) and TUNEL (fragmented DNA; red) and Hoechst nuclear stain (blue) in BMDM treated with 40 μ l/ml BCM or 10 μ g/ml CIRP for 24 h. ( f ) Confocal immunofluorescence images and analysis of colocalization of LC3 (autophagosome; green) and TUNEL (fragmented DNA; red) and Hoechst nuclear stain (blue) in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μ l/ml) for 24 h. ( g ) Confocal immunofluorescence images showing colocalization of mitochondria (MitoTracker; green) and lysosomes (LysoTracker; red) in BMDM, or J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μ l/ml) for 24 h. ( h ) Expression levels of p62 mRNA or ( i ) protein expression in BMDM stimulated by BCM (40 μ l/ml) for 0–24 h. ( j ) Western blot of whole-cell lysates showing p62 protein expression in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, then stimulated with BCM (40 μ l/ml) for 0–6 h. ( k ) Confocal immunofluorescence images and analysis of colocalization of mitochondria (MitoTracker; green), LC3(autophagosomes; red) and p62 (cyan) in BMDM stimulated with BCM (40 μ l/ml) or CIRP (10 μ g/ml) for 24 h. Arrows indicate the colocalization of mitochondria-LC3-p62 (white). All results are representative of three independent experiments. The graphs show the mean and S.E.M., n =3. Significances between groups were determined by using independent samples two-tailed Student’s t -test. * P <0.05 and ** P <0.01 versus the control or compared between the indicated groups

Article Snippet: Primary antibodies for cell staining were: EEA1 (endosomal marker) antibody (2411S, Cell Signaling Technology, Danvers, MA, USA), LC3 antibody (4599S, Cell Signaling Technology), RIPK1 antibody (610458, BD Biosciences, San Jose, CA, USA), RIPK3 antibody (sc-135170, Santa Cruz Biotechnology, Dallas, TX, USA), MitoTracker and LysoTracker (M7514 and L7528, Thermo Fisher Scientific, Pittsburgh, PA, USA), p62/SQSTM1 Antibody (MAB8028, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, TUNEL Assay, Staining, Two Tailed Test

Journal: Molecular neurobiology

Article Title: Citalopram reduces aggregation of ATXN3 in a YAC transgenic mouse model of Machado-Joseph disease

doi: 10.1007/s12035-018-1331-2

Figure Lengend Snippet: Assessment of macroautophagy markers shows increased levels of p62 in the spinal cord (B), and of p62 and beclin-1 in the cerebellum (C) of citalopram-treated mice compared to controls. Citalopram treatment did not affect levels of LC3 in any of the three regions (A-C). (A, B, and C) Left panels show immunoblots of indicated proteins in soluble fractions of brainstem, spinal cord and cerebellum. Right panels display quantification of band intensity, with values normalized to Gapdh. Bars represent the average percentage of protein relative to vehicle-treated mice (± SEM). Statistical significance is indicated as *P < 0.05 and **P < 0.01. Mice 1, 2, and 20 were excluded from statistical analysis. Diamond (◆) represents p62 bands.

Article Snippet: Total protein lysates from soluble fractions (100 μg from brainstem, and cerebellum) were resolved on 10% SDS-PAGE gels, and corresponding PVDF membranes were incubated overnight at 4 °C with primary antibodies: mouse anti-HSP70 (1:500; SPA810, Enzo Life Sciences), rabbit anti-HSP90α (1:1000; ab2928, Abcam), mouse anti-HSP90β (1:1000; ADI-SPA842, Enzo Life Sciences), rabbit anti-HSP40 (1:1000; #4868, Cell Signaling), mouse anti-p62/SQSTM1 (1:1000; H00008878-M01, Novus Biologicals), rabbit anti-Beclin1 (1:1000; ab207612, Abcam), rabbit anti-LC3 (1:500; PM036, MBL International Corporation), mouse anti-GAPDH (1:5000; MAB374, Millipore), and rabbit anti-MJD [ 30 ] (1:30000).

Techniques: Western Blot

Journal: Cell Death and Differentiation

Article Title: The generation of neutrophils in the bone marrow is controlled by autophagy

doi: 10.1038/cdd.2014.169

Figure Lengend Snippet: Lack of Atg5 mRNA and ATG5 protein expression as well as a functional defect in autophagy in neutrophils of Atg5NΔ mice. (a) Quantitative real-time PCR. Freshly purified immature and mature bone marrow-derived neutrophil samples from WT and Atg5NΔ mice were analyzed for Atg5 expression. S18 was used as a reference gene to normalize the expression of Atg5. Values are means±S.E.M. of two independent experiments. (b) Immunoblotting. Freshly purified mature bone marrow-derived neutrophils of WT and Atg5NΔ mice were analyzed for ATG5 protein expression. The neutrophil-depleted bone marrow samples were lysed and immunoblotted next to the neutrophil lysates to demonstrate the cell specificity of the ATG5 knockout. If present, ATG5 protein is seen as a conjugate with ATG12. The accumulation of p62 confirms the attenuated autophagic activity in ATG5-deficient neutrophils. The results are representative of three independent experiments. The quantification of the ATG5 signal was performed using the Odyssey Fc Imaging System (LI-COR Biosciences GmbH, Bad Homburg, Germany) and the image Studio software. (c) Confocal microscopy. Freshly purified mature bone marrow-derived neutrophils of WT and Atg5NΔ mice were cultured in the presence and absence of rapamycin for 1 h and stained with AUTOdot autophagy visualization dye. The dye is specific for autophagic vacuoles that can be detected in rapamycin-treated WT neutrophils as bright white dots. In contrast, rapamycin had no detectable effect on neutrophils derived from Atg5NΔ mice. Bars, 10 μm. (Right) Statistical analysis of three independent confocal microscopy experiments. Values are means±S.E.M.

Article Snippet: Antibodies The antibodies used for immunoblotting were polyclonal rabbit anti-ATG5 (1 : 500; Novus Biologicals, LuBioScience GmbH, Lucerne, Switzerland), polyclonal rabbit anti-p62 (1 : 4000; Novus Biologicals), monoclonal mouse anti-LC3 (1 : 500; Nanotools, Teningen, Germany), monoclonal mouse anti-Beclin 1 (1 : 1000; Abgent, LuBioScience GmbH), polyclonal rabbit anti-MMP-9 (1 : 1000; Abcam, Cambridge, UK), and monoclonal mouse anti-GAPDH (1 : 5000; Millipore, Bedford, MA, USA).

Techniques: Expressing, Functional Assay, Real-time Polymerase Chain Reaction, Purification, Derivative Assay, Western Blot, Knock-Out, Activity Assay, Imaging, Software, Confocal Microscopy, Cell Culture, Staining

Journal: Cell Death and Differentiation

Article Title: The generation of neutrophils in the bone marrow is controlled by autophagy

doi: 10.1038/cdd.2014.169

Figure Lengend Snippet: Downregulation of ATG5 expression in immature neutrophil precursors results in accelerated differentiation. (a) Quantitative real-time PCR. Undifferentiated Hoxb8 neutrophils were transduced with virus encoding control or Atg5-specific shRNA, and subsequently analyzed. S18 was used as a reference gene to normalize the expression of Atg5. Values are means±S.E.M. of three independent experiments. (b) Immunoblotting. ATG5 expression is decreased in Atg5 shRNA-treated undifferentiated Hoxb8 neutrophils. Monomeric ATG5 was not detectable in these cells. The reduced presence of the ATG5-ATG12 conjugate was correlated with reduced LC3-II and higher p62 levels, indicating attenuated autophagic activity in these cells. Results are representative of three independent experiments. (c) Light microscopy. Undifferentiated Hoxb8 neutrophils were treated with control or Atg5 shRNA and then allowed to differentiate for 4 days. Cytospins were stained with the Hematocolor Set and quantified. Based on cell size and nuclear morphology, cells were categorized as myelocytes, metamyelocytes, band cells, or segmented neutrophils. Values are means±S.E.M. of three independent experiments. (d) Flow cytometry. Undifferentiated Hoxb8 neutrophils were treated with control and Atg5 shRNA, allowed to differentiate for 3 days, and Gr-1 surface expression analyzed. Values are means±S.E.M. of four independent experiments. (Right) Representative original flow cytometric data are shown. Red, Gr-1 expression. Blue, isotype-specific control antibody staining. (e) Immunoblotting. Undifferentiated Hoxb8 neutrophils were treated with control and Atg5 shRNA, allowed to differentiate for 4 days, and MMP-9 expression analyzed. Results are representative of three independent experiments. See Supplementary Figure S6 for the characterization of the Hoxb8 neutrophil differentiation system

Article Snippet: Antibodies The antibodies used for immunoblotting were polyclonal rabbit anti-ATG5 (1 : 500; Novus Biologicals, LuBioScience GmbH, Lucerne, Switzerland), polyclonal rabbit anti-p62 (1 : 4000; Novus Biologicals), monoclonal mouse anti-LC3 (1 : 500; Nanotools, Teningen, Germany), monoclonal mouse anti-Beclin 1 (1 : 1000; Abgent, LuBioScience GmbH), polyclonal rabbit anti-MMP-9 (1 : 1000; Abcam, Cambridge, UK), and monoclonal mouse anti-GAPDH (1 : 5000; Millipore, Bedford, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transduction, Virus, Control, shRNA, Western Blot, Activity Assay, Light Microscopy, Staining, Flow Cytometry

Journal: Cell Death and Differentiation

Article Title: The generation of neutrophils in the bone marrow is controlled by autophagy

doi: 10.1038/cdd.2014.169

Figure Lengend Snippet: Ectopic Atg5 expression in immature neutrophil precursors results in delayed differentiation. (a) Quantitative real-time PCR. Undifferentiated Hoxb8 neutrophils were subjected to lentiviral transduction with control vector or Atg5 gene construct, and subsequently analyzed. S18 was used as a reference gene to normalize the expression of Atg5. Values are means±S.E.M. of three independent experiments. (b) Immunoblotting. Atg5 gene transfer results in strong expression of the 33-kDa ATG5 monomer and slightly increased expression of ATG5 within the conjugate with ATG12. Ectopic ATG5 expression was associated with higher LC3-II and reduced p62 levels, suggesting increased autophagic flux in these cells. Results are representative of three independent experiments. (c) Light microscopy. Undifferentiated Hoxb8 neutrophils were subjected to lentiviral control vector and Atg5 gene transfer and then allowed to differentiate for 4 days. Cytospins were stained with the Hematocolor Set and quantified. Based on cell size and nuclear morphology, cells were categorized as myelocytes, metamyelocytes, band cells, or segmented neutrophils. Values are means±S.E.M. of three independent experiments. (d) Flow cytometry. Undifferentiated Hoxb8 neutrophils were subjected to lentiviral control vector and Atg5 gene transfer, allowed to differentiate for 3 days, and the Gr-1 expression analyzed. Values are means±S.E.M. of four independent experiments. (Right) Representative original flow cytometric data are shown. Red, Gr-1 expression. Blue, isotype-matching control antibody. (e) Immunoblotting. Undifferentiated Hoxb8 neutrophils were subjected to lentiviral control vector and Atg5 gene transfer, allowed to differentiate for 4 days, and MMP-9 expression analyzed. Results are representative of three independent experiments. See Supplementary Figure S6 for the characterization of the Hoxb8 neutrophil differentiation system

Article Snippet: Antibodies The antibodies used for immunoblotting were polyclonal rabbit anti-ATG5 (1 : 500; Novus Biologicals, LuBioScience GmbH, Lucerne, Switzerland), polyclonal rabbit anti-p62 (1 : 4000; Novus Biologicals), monoclonal mouse anti-LC3 (1 : 500; Nanotools, Teningen, Germany), monoclonal mouse anti-Beclin 1 (1 : 1000; Abgent, LuBioScience GmbH), polyclonal rabbit anti-MMP-9 (1 : 1000; Abcam, Cambridge, UK), and monoclonal mouse anti-GAPDH (1 : 5000; Millipore, Bedford, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transduction, Control, Plasmid Preparation, Construct, Western Blot, Light Microscopy, Staining, Flow Cytometry

Journal: Cell Death and Differentiation

Article Title: The generation of neutrophils in the bone marrow is controlled by autophagy

doi: 10.1038/cdd.2014.169

Figure Lengend Snippet: Autophagic activity declines during neutrophil differentiation and pharmacological induction of autophagy delays the process of neutrophil differentiation. (a) Immunoblotting. Hoxb8 neutrophil progenitors were allowed to differentiate and analyzed at the indicated times (see Supplementary Figure S6 for the characterization of the Hoxb8 neutrophil differentiation system). The autophagic activity appeared to decline at later time points as indicated by reduced LC3-II and higher p62 levels. ATG5 and Beclin 1 expression did not change during the process of neutrophil differentiation. However, phosphorylation (Thr389) of p70S6K increased, suggesting increased mTORC1 activity. Results are representative of four independent experiments. (b) Immunoblotting. Hoxb8 neutrophil progenitors were allowed to differentiate in the presence and absence of 100 nM rapamycin and analyzed 3 days after initiation of the differentiation process. Rapamycin treatment resulted in higher LC3-II and reduced p62 levels, suggesting induction of autophagy. The results are representative of two independent experiments. (c) Flow cytometry. Hoxb8 neutrophil progenitors were allowed to differentiate in the presence and absence of 100 nM rapamycin and analyzed 3 days after initiation of the differentiation process. Rapamycin treatment resulted in lower Gr-1 surface expression, suggesting a delayed differentiation process as a consequence of autophagy induction. Representative original flow cytometric data are shown. Red, Gr-1 expression. Blue, isotype-matching control antibody. The indicated values are mean Gr-1/control MFI ratios±S.E.M. of four independent experiments

Article Snippet: Antibodies The antibodies used for immunoblotting were polyclonal rabbit anti-ATG5 (1 : 500; Novus Biologicals, LuBioScience GmbH, Lucerne, Switzerland), polyclonal rabbit anti-p62 (1 : 4000; Novus Biologicals), monoclonal mouse anti-LC3 (1 : 500; Nanotools, Teningen, Germany), monoclonal mouse anti-Beclin 1 (1 : 1000; Abgent, LuBioScience GmbH), polyclonal rabbit anti-MMP-9 (1 : 1000; Abcam, Cambridge, UK), and monoclonal mouse anti-GAPDH (1 : 5000; Millipore, Bedford, MA, USA).

Techniques: Activity Assay, Western Blot, Expressing, Phospho-proteomics, Flow Cytometry, Control

Journal: Cell Death and Differentiation

Article Title: The generation of neutrophils in the bone marrow is controlled by autophagy

doi: 10.1038/cdd.2014.169

Figure Lengend Snippet: Activity of p38 increases during neutrophil differentiation and its pharmacological inhibition induces autophagy and reduces the process of neutrophil differentiation. (a) Immunoblotting. Hoxb8 neutrophil progenitors were allowed to differentiate and analyzed at the indicated times (see Supplementary Figure S6 for the characterization of the Hoxb8 neutrophil differentiation system). Phosphorylation (Thr180/Tyr182) of p38 increased, suggesting increased p38 activity. On the other hand, ERK1/2 was completely dephosphorylated (Thr202/Thr204) during terminal differentiation. Results are representative of four independent experiments. We obtained no evidence for a role for the PI3K pathway in neutrophil differentiation (see Supplementary Figure S9). (b) Immunoblotting. Hoxb8 neutrophil progenitors were allowed to differentiate in the presence and absence of 1 μM PD169316 (inhibitor of p38), and analyzed 3 days after initiation of the differentiation process. PD169316 treatment resulted in higher LC3-II and lower p62 levels, indicating induction of autophagy. The results are representative of two independent experiments. (c) Flow cytometry. Hoxb8 neutrophil progenitors were allowed to differentiate in the presence and absence of 1 μM PD169316 (inhibitor of p38) or 1 μM PD98059 (inhibitor of ERK1/2), and analyzed 3 days after initiation of the differentiation process. PD169316 treatment resulted in lower Gr-1 surface expression, suggesting a delayed differentiation process as a consequence of autophagy induction. In contrast, pharmacological inactivation of ERK1/2 had no effect in this system. Values are means±S.E.M. of four independent experiments. Representative original flow cytometric data are shown below. Red, Gr-1 expression. Blue, isotype-matching control antibody. (d) Immunoblotting. Hoxb8 neutrophil progenitors were allowed to differentiate in the presence and absence of 1 μM PD169316 (inhibitor of p38), and analyzed 3 days after initiation of the differentiation process. PD169316 treatment resulted in decreased phosphorylation (Thr389) of p70S6K, suggesting diminished mTORC1 activity. Results are representative of two independent experiments

Article Snippet: Antibodies The antibodies used for immunoblotting were polyclonal rabbit anti-ATG5 (1 : 500; Novus Biologicals, LuBioScience GmbH, Lucerne, Switzerland), polyclonal rabbit anti-p62 (1 : 4000; Novus Biologicals), monoclonal mouse anti-LC3 (1 : 500; Nanotools, Teningen, Germany), monoclonal mouse anti-Beclin 1 (1 : 1000; Abgent, LuBioScience GmbH), polyclonal rabbit anti-MMP-9 (1 : 1000; Abcam, Cambridge, UK), and monoclonal mouse anti-GAPDH (1 : 5000; Millipore, Bedford, MA, USA).

Techniques: Activity Assay, Inhibition, Western Blot, Phospho-proteomics, Flow Cytometry, Expressing, Control

Journal: Arthritis Research & Therapy

Article Title: Association between autophagy and inflammation in patients with rheumatoid arthritis receiving biologic therapy

doi: 10.1186/s13075-018-1763-0

Figure Lengend Snippet: Representative cytometric histograms of p62 levels in lymphocytes ( a 1 and b 1), monocytes ( a 2 and b 2), and granulocytes ( a 3 and b 3) from one patient with rheumatoid arthritis (RA) and one healthy control subject (HC). Gray shadows indicate cytometric histograms of the stained p62 expression in immune cells from patients with RA and HC. Comparisons of p62 mean fluorescence intensity in lymphocytes ( c ), monocytes ( d ), and granulocytes ( e ) between patients with RA and HC. Data are presented as box plot diagrams, with the box encompassing the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates the median value for each group. * p < 0.05, ** p < 0.001 vs. HC

Article Snippet: Cells were fixed by adding 100 μl of reagent 1 (Beckman Coulter Life Sciences) for 15 min and were centrifuged for 5 min at 300 × g . After removal of the supernatant, 100 μl of reagent 2 (Beckman Coulter Life Sciences) was added for permeabilization for 10 min, and cells were subsequently incubated with PerCP-conjugated p62/SQSTM1 mAb (clone 5H7E2; Novus Biologicals, Littleton, CO, USA) for 15 min in the dark at RT.

Techniques: Control, Staining, Expressing, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Association between autophagy and inflammation in patients with rheumatoid arthritis receiving biologic therapy

doi: 10.1186/s13075-018-1763-0

Figure Lengend Snippet: Correlation between autophagy expression and inflammatory parameters in 72 patients with rheumatoid arthritis

Article Snippet: Cells were fixed by adding 100 μl of reagent 1 (Beckman Coulter Life Sciences) for 15 min and were centrifuged for 5 min at 300 × g . After removal of the supernatant, 100 μl of reagent 2 (Beckman Coulter Life Sciences) was added for permeabilization for 10 min, and cells were subsequently incubated with PerCP-conjugated p62/SQSTM1 mAb (clone 5H7E2; Novus Biologicals, Littleton, CO, USA) for 15 min in the dark at RT.

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: Association between autophagy and inflammation in patients with rheumatoid arthritis receiving biologic therapy

doi: 10.1186/s13075-018-1763-0

Figure Lengend Snippet: Representative example of LC3-II/LC3-I and p62 protein expression in peripheral blood mononuclear cell lysates from one patient with rheumatoid arthritis (RA) and one healthy control subject (HC) ( a ). Comparisons of protein expression levels of LC3-II/LC3-I ( b ) and p62 ( c ) in patients with RA and HC are shown. Data are presented as box plot diagrams, with the box encompassing the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates median value for each group. * p < 0.005, ** p < 0.001 versus HC, determined by Mann-Whitney U test

Article Snippet: Cells were fixed by adding 100 μl of reagent 1 (Beckman Coulter Life Sciences) for 15 min and were centrifuged for 5 min at 300 × g . After removal of the supernatant, 100 μl of reagent 2 (Beckman Coulter Life Sciences) was added for permeabilization for 10 min, and cells were subsequently incubated with PerCP-conjugated p62/SQSTM1 mAb (clone 5H7E2; Novus Biologicals, Littleton, CO, USA) for 15 min in the dark at RT.

Techniques: Expressing, Control, MANN-WHITNEY