Journal: bioRxiv

Article Title: Physicochemical characterization, toxicity and in vivo biodistribution studies of a discoidal, lipid-based drug delivery vehicle: Lipodisq nanoparticles containing doxorubicin

doi: 10.1101/2020.06.18.159087

Figure Lengend Snippet: A. LQ-10% (1:25) or free DOX (13.6 μM) was incubated in PBS pH 7.4 or 5.0 at 37 °C for 4 h. Then half of the solution was filtered through a Centrifree ® spin column to separate free DOX from Lipodisq-bound drug. Both the filtrate and the unfiltered half were diluted further (1:40) in complete medium and added to HeLa cells. Untreated LQ-10% was added as a control. The cells were incubated for 24 h before the level of thymidine incorporation was determined by incubation with [ 3 H]thymidine for 30 min. The data were normalised to untreated control cells. B. HeLa cells were treated with the vATPase inhibitors Bafilomycin A1 (BafA1, 25 nM) or Concanamycin A (ConA, 25 nM) for 1 h, then the acid-sensitive lysosomal stain Lysotracker Green (100 nM) and the nuclear stain Hoechst (1.6 μM) were added and images were acquired after 5 min. Scale bar; 10 μm. C. HeLa cells were treated with increasing concentrations of LQ-10% for 24 h in the absence or presence of BafA1 (50 nM) or ConA (40 nM). Then thymidine incorporation was assessed, and the data were normalised to non Lipodisq-treated cells for each series. The graphs show mean values ± SEM from three independent experiments except for free DOX in A, where the mean values from two independent experiments are shown and the error bars show deviation from the mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Bafilomycin A1 (BafA1) was from Enzo Life Sciences, Farmingdale, NY.

Techniques: Incubation, Staining

Journal: Nature

Article Title: Stress granules plug and stabilize damaged endolysosomal membranes

doi: 10.1038/s41586-023-06726-w

Figure Lengend Snippet: a , Representative images of iPSDM left untreated or treated with NaAsO 2 (0.5 mM, 1 h) and stained for G3BP1 (magenta) and GAL-3 (green). b , Bar plots show quantification of G3BP1, and GAL-3 puncta area normalised to cell area of iPSDM treated as in (a). Data represent the mean ± SEM of at least 10 cells from one out of three independent experiments ( n = 30 cells examined over three independent experiments, two-tailed t-test). c , immunoblot for phospho- (p) and total levels of eIF2a in iPSDM left untreated or treated with NaAsO 2 (0.5 mM) or LLOMe (1 mM) at the indicated time points. ACTB was used as a loading control. d , shows eIF2a protein levels relative to ACTB, Bar plot represents mean values ± SEM ( n = three independent experiments). e , Representative images of iPSDM left untreated or treated with LLOMe (1 mM, 20 min) and incubated in the presence or absence of 10 µg/ml cycloheximide (CHX) or 10 µg/ml emetine; and stained for G3BP1 (top panel) and GAL-3 (bottom panel). f , Bar plots show high-content imaging quantification of G3BP1, and GAL-3 puncta area normalised to cell area of iPSDM treated as in (e). Data represent the mean ± SEM ( n = three independent experiments). P -value was calculated using one-way ANOVA, Tukey’s multiple comparisons test. g , iPSDM were incubated in the presence or absence of BAFA1 (100 nM, 1 h) and left untreated or treated with silica crystals (200 µg/mL, 3 h), and stained for GB3P1 and GAL-3. Silica crystals were imaged using confocal reflection microscopy. h , Bar plot shows the quantification of G3BP1 puncta area normalised to cell area. Data represent the mean ± SEM of at least 10 cells from one out of three independent experiments ( n = 30 cells examined over 3 independent experiments) P -value was calculated using one-way ANOVA, Tukey’s multiple comparisons test. i , j , Fixed image analysis of G3BP1-LAMP1 (lysosome) interactions (i) in iPSDM stimulated with LLOMe (1 mM, 30 min). #1–3 show three different z-stack sequences corresponding to the cell regions indicated in (j) (red squares). Arrowheads illustrate different membrane and intralumenal G3BP1-LAMP1 interactions. k , Bar plots show G3BP1/LAMP1 SRCC and the percentage of events where G3BP1 associates with LAMP-1 + structures. Data represent the mean ± SEM of at least 10 cells from one out of 3 independent experiments ( n = 30 cells examined over 3 independent experiments). Scale bar: 10 μm, zoom-in: 2 μm. For gel source data, see Supplementary Fig. .

Article Snippet: In experiments with BAFA1 (Merck, B1793-10UG), iPSDM were pre-incubated for 1 h with a 100 nM solution.

Techniques: Staining, Two Tailed Test, Western Blot, Incubation, Imaging, Microscopy, Membrane

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: BafA1 treatment modulates aggregate formation and levels of extracellular SNCA in transfected H4 cells. (A) to (C): Immunocytochemistry of H4 cells transfected with SNCA, SNCA-T and SNCA-T and SNCAIP: (A) Large aggregates of different size and morphology can be observed after transfection with high-aggregating SNCA-T and SNCAIP whereas SNCA only occasionally leads to smaller intracellular aggregates sized below 1 μm. Scale bar: 5 μm. (B) Quantification of differentially sized aggregates per cell in H4 cells transfected with SNCA-T alone compared to cotransfection of SNCA-T with SNCAIP. (C) Treatment with 200 nM BafA1 for 12 h results in smaller and more punctate intracellular structures. (D) to (F): Dot blot analysis of extracellular SNCA in the conditioned medium of H4 neuron-like cells and CG4 oligodendroglial cells: (D) Representative dot blots of H4 cell medium containing extracellular SNCA. Recombinant SNCA is used as quantification standard. (E) Dot blot quantification of extracellular SNCA levels in similar volumes of conditioned medium of H4 cells expressing high-aggregating SNCA-T compared to low-aggregating SNCA and mock-transfected control cells 36 h post-transfection. Treatment with 200 nM BafA1 for 12 h results in slightly increased levels of extracellular SNCA in the medium of H4 cells transfected with SNCA, and both SNCA-T and SNCAIP compared to untreated cells. (F) Representative dot blots showing a lack of extracellular SNCA in supernatants of undifferentiated and 6 d differentiated rat oligodendroglial CG4 cells overexpressing SNCA to exclude nonspecific SNCA release. All values are presented as mean + s.e.m; (*) P = 0.009.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transfection, Immunocytochemistry, Cotransfection, Dot Blot, Recombinant, Expressing

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Quantification of aggregates in transfected H4 cells

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transfection, Construct

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Enhanced apoptotic action in the microenvironment of SNCA-expressing H4 cells, by BafA1 treatment. (A) to (D): Microenvironmental analysis of activated CASP3 as measure for apoptosis 36 h post-transfection of H4 cells with SNCA, SNCA-T, or SNCA-T and SNCAIP by immunocytochemical quantification. (A) Treatment with BafA1 increases CASP3+ cells for all constructs in the transient transfection model. (B) Quantification of CASP3+ and SNCA+ cells for all constructs in the transient transfection model. (C) Left: Cartoon of the analysis paradigm of the microenvironmental toxicity: SNCA-positive fields (white outlined, “microenvironment”) are defined by the presence of a nuclei (blue) of SNCA-overexpressing cell (green). SNCA-negative fields (gray outlined) do not contain nuclei of SNCA-overexpressing cells and are referred to as “distant” fields. The number of CASP3+ cells (red) within SNCA-positive and SNCA-negative fields are quantified 36 h post-transfection of H4 cells with SNCA, SNCA-T, or SNCA-T and SNCAIP. Depicted fields are part of a grid (6 × 7 fields) which is based on the mean number of SNCA-positive cells (≈ 21 SNCA+ cells/image; 20 × magnification) for all transfected constructs. Right: Representative immunocytochemical stainings for all transfected constructs displaying the grid. Scale bar 20 μm. (D) Quantification of CASP3+ and SNCA− H4 cells within the microenvironment of SNCA, SNCA-T or SNCA-T and SNCAIP-overexpressing cells with and without BafA1 treatment for 12 h. All values are mean + s.e.m; (*) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Expressing, Transfection, Construct

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Activation of CASP3 in the microenvironment of H4 cells expressing low- and high-aggregating aSyn

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Activation Assay, Expressing

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: (See previous page). Characterization of particle fractions (PFs) prepared from conditioned medium of transfected H4 cells. (A) to (F) Western blot and dot blot analysis of SNCA associated with PFs prepared from conditioned medium of H4 cells expressing high-aggregating SNCA-T and SNCAIP or low-aggregating SNCA. (A) Representative protein gel blots of total SNCA+ PFs prepared from 12 ml conditioned medium of transfected H4 cells -/+BafA1 show an increased presence of extracellular SNCA (14 kDa) or SNCA-T (27 kDa) after BafA1 treatment. (B) 12 ml supernatant after PF separation contain only low levels of soluble SNCA. (C) Representative western blots of SNCA PFs -/+BafA1 after filter retardation to exclude particles of different size by using distinct pore size filters reveal enlarged particles (>1 .2 μm) in the presence of BafA1. (D) Representative dot blots of SNCA in the conditioned medium of H4 cells expressing low-aggregating SNCA -/+BafA1. (E) Representative protein gel blots of SNCA-T and SNCAIP PFs after exclusion of particles of different size by using distinct pore size filters. (F) Quantification of SNCA associated with PFs after using filters of defined pore size normalized to SNCA levels in unfiltered PFs displaying a shift toward smaller particles by BafA1. (G) BafA1 effect on oligomerization analysis of SNCA associated with PFs prepared from H4 cells expressing SNCA, as well as SNCA-T and SNCAIP measured by sucrose gradient centrifugation. All values are mean + s.e.m. Differences are significant at (*) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transfection, Western Blot, Dot Blot, Expressing, Gradient Centrifugation

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Toxic response of naïve H4 cells exposed to extracellular SNCA. (A) Immunocytochemistry of CASP3+ cells after 6 h exposure of naïve H4 cells to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, and both SNCA-T and SNCAIP (and control H4 cells). Scale bar 40 μm. (B) Increase of toxicity in naïve H4 cells exposed to PFs containing extracellular SNCA from untreated and BafA1-treated cells after 6 h compared to exposure with control medium measured by AK release using ToxiLight assay. (C) Higher magnification of CASP3+ cells reveals an association to the surface of exposed H4 cells and intracellular accumulation of SNCA+ PFs. Scale bar 5 μm. All values are mean + s.e.m; differences were significant at (*) P < 0.05 and (#) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Immunocytochemistry, Transfection

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Toxic response of primary neuronal cultures exposed to extracellular SNCA. (A) Exposure of primary hippocampal neuronal cultures (TUBB3, neuronal marker; GFAP, astroglial marker) to extracellular SNCA associated with particle fractions (PFs) prepared from conditioned medium of H4 cells transfected with SNCA, SNCA-T, SNCA-T and SNCAIP (and control H4 cells). (B) The toxic response of primary neurons exposed to PFs from untreated and BafA1-treated H4 cells after 6 h exposure time measured by AK release using ToxiLight assay. (C) Exposure of primary neuronal cultures to SNCA, as well as SNCA-T and SNCAIP PFs leads to the formation of intracellular SNCA+ accumulations in TUBB3+ neuronal cells. Z-stack of confocal images, scale bar 10 μm. All values are mean + s.e.m. Differences were significant at (*) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Marker, Transfection

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: (See previous page). Modulation of intracellular and extracellular SNCA in transgenic mice, by BafA1. (A) Immunohistochemistry of transgenic mice overexpressing human SNCA under the control of the PDGFB-promoter (SNCA-tg) and nontransgenic mice (non-tg) using an antibody recognizing human and mouse SNCA. Transgenic mice display SNCA+ inclusions in neuronal bodies and in the neuropil of the neocortex (Neoctx) compared to non-tg mice. BafA1 has been applied systemically by daily intraperitoneal injection of 0.3 mg/kg or vehicle (saline) for 5 consecutive days. The inset indicates the representative region of neocortex depicted in the 2nd and in the 3rd row. Scale bar 200 μm. Transgenic mice display SNCA immunoreactivity in the molecular layer (ML) of the hippocampus (Hippo) indicated by black arrows. Scale bar 50 μm. CA1: cornu ammonis field1; SL: stratum lacunosum (B) BafA1 treatment of SNCA-tg mice diminishes the number of neurons bearing SNCA+ inclusions within the neocortex compared to vehicle-treated SNCA-tg mice. (C) Quantification of the percentage of the neocortical neuropil area that shows SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. (D) Percentage of the neuropil area in the hippocampal ML that displays SNCA immunoreactivity in SNCA-tg mice after BafA1 treatment. (E) Left: Representative western blots of protein lysates probed for endogenous mouse and human SNCA depict monomeric (14 kDa) and oligomeric species (42 and 56 kDa). ACTB serves as loading control (42 kDa). Right: Western blot quantification of SNCA expression levels in BafA1-treated transgenic mice overexpressing human SNCA and normalized to ACTB (n = 3 animals per group). (F) Extracellular SNCA levels in the CSF of transgenic mice overexpressing SNCA measured by ELISA. All values are mean + s.e.m. Differences are significant at (#) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transgenic Assay, Immunohistochemistry, Injection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Neuronal loss paralleled by astro- and microgliosis in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of neuronal loss and astrogliosis paralleled by a microgliosis. (A) Representative images of RBFOX3/NeuN+ neurons, GFAP+ astroglia and the microglial marker AIF1 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. The insets in the 1st row depict the region of quantification. Insets in the 2nd row depict regions which are magnified in the 3rd row. Scale bar for RBFOX3/NeuN in the hippocampus 50 μm, GFAP in the hippocampus 50 μm and 20 μm (2nd and 3rd row) and AIF1 in the hippocampus and the neocortex 20 μm (4th and 5th row). Quantification of cell number and optical density quantification of (B) the neuronal marker RBFOX3/NeuN+, (C) the astroglial marker GFAP, (D) the microglial marker AIF1 in the hippocampus, and (E) AIF1 in the neocortex of SNCA-tg mice compared to non-tg mice. (B) to (E) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. All values are mean + s.e.m; differences are significant at (#) P < 0.05, unpaired t test, and at (*) P < 0.01.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transgenic Assay, Immunohistochemical staining, Marker

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Inflammatory response in transgenic mice, by BafA1 treatment. Immunohistochemical assessment of the inflammatory response in SNCA transgenic mice. (A) Representative images of the inflammatory marker TNF and IL6 in the hippocampus of SNCA-tg and non-tg mice treated with BafA1 or vehicle. Black arrows indicate TNF and IL6 immunoreactivity. Scale bar for TNF and IL6 in the hippocampus 30 μm. Optical density quantification of (B) the inflammatory markers TNF and (D) IL6 in the hippocampus of SNCA-tg mice compared to non-tg mice. (B) to (D) Effects of BafA1 treatment in the hippocampus of SNCA-tg mice compared to vehicle treatment. (C) The absolute amount of TNF as measured by ELISA in SNCA-tg mice treated with BafA1 compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P < 0.05, unpaired t test and at (*) P < 0.01.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Transgenic Assay, Immunohistochemical staining, Marker, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: BafA1 treatment increases the accumulation of SNCA in glial cells in the microenvironment of SNCA-expressing neurons in transgenic mice. Immunohistochemical analysis of glial cells (S100B+) in the microenvironment of SNCA+ neurons within the colliculus and the hippocampus of SNCA-tg mice. (A) SNCA immunoreactivity in glial cells in the colliculus of SNCA-tg mice compared to non-tg mice -/+BafA1 treatment. Insets in the 1st row indicate the region within the superior colliculus which is magnified in the 2nd row. Black arrowheads indicate the presence of SNCA+ neurons based on their morphological appearance in close proximity. Scale bar 1st row 50 μm, 2nd and 3rd row 20 μm. (B) Quantification of glial cells based on their morphology displaying SNCA immunoreactivity in the colliculus of SNCA-tg mice -/+BafA1 treatment. (C) Confocal images of S100B+ astroglia (red) double-labeling for SNCA (green) in close proximity to SNCA+ neurons (N; defined by morphology) in the hippocampus and colliculus of SNCA-tg mice either treated with BafA1 or vehicle. (D) and (E) Quantification of S100B+ and SNCA+ cells after BafA1 treatment in SNCA-tg mice compared to vehicle-treated animals. All values are mean + s.e.m; differences are significant at (#) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Expressing, Transgenic Assay, Immunohistochemical staining, Labeling

Journal: Autophagy

Article Title: Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

doi: 10.4161/auto.36436

Figure Lengend Snippet: Modulation of nonclassical secretory pathway by BafA1 treatment in H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T. (A) Scheme of markers for distinct nonclassical secretory pathways including exosome release, endocytic recycling pathway, and microparticle shedding. MVBs = multivesicular bodies, ILVs = intraluminal vesicles, PS = phosphatidylserine (B) Quantification of extracellular CD63 levels in the medium of H4 cells expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP compared to control cells (mock) by dot blot analysis. Cells have been treated with or without 200 nM BafA1 for 12 h. (C) Quantification of RAB11A expression in H4 cell lysates expressing low-aggregating SNCA and high-aggregating SNCA-T and SNCAIP -/+ BafA1 treatment. (D) Left: FACS analysis of released microparticles by transfected H4 cells -/+ BafA1 using a FITC-labeled ANXA5 antibody. Right: Representative scatter plots and histograms showing medium with ANXA5 as control for background signals, unstained microparticles to exclude auto fluorescence and microparticles stained for ANXA5 as positive control defining gating criteria. All values are mean + s.e.m; differences are significant at (*) P < 0.01 and at (#) P < 0.05.

Article Snippet: Cells were treated 24 h post-transfection with 200 nM BafA1 (Applichem, A7823,0001), 5 mM 3-methyladenine (3-MA, Sigma Aldrich, M9281), 50 μM chloroquine diphosphate salt (CQ, Sigma Aldrich, C6628), or vehicle (DMSO or aqua dest.) for 12 h and fixed or harvested for further analysis.

Techniques: Expressing, Dot Blot, Transfection, Labeling, Fluorescence, Staining, Positive Control

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: Experiments supplementary to Figs. 1 and ​and22. (a and b) Primary HDFs (a) and primary MEFs (b) were treated with 60 μg/ml cGAMP, and cell lysates were collected at the indicated time points. (c) WT HeLa cells were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 and with or without wortmannin (200 nM) or VPS34-IN1 (300 nM) for 4 h. All the following experiments were treated the same unless specified. (d) MEFs were treated with 60 μg/ml cGAMP alone and with wortmannin for 2 h. All the following experiments using MEFs use the same conditions. (e and f) WT, WIPI2 KO (e), and WIPI 4KO (f) HeLa cells stably expressing GFP-STING were incubated in starvation media alone and with BafA1 for 4 h. (g) MEFs were incubated in starvation media alone and with BafA1 for 4 h. (h and i) MEFs were incubated with cGAMP alone and with BafA1 (h) or with BafA1 and wortmannin (i). (j) WT and FIP200 KO HeLa cells were incubated with 15 µg/ml cGAMP alone and with 20 μM chloroquine (CQ) for 8 h. (k) Representative Airyscan-processed confocal imaging of FIP200 KO HeLa cells with stable expression of RFP-LC3B and GFP-STING treated with cGAMP (60 µg/ml) for 8 h, fixed, and immunostained for endogenous LAMP2. Scale bar, 10 µm. Western blotting experiments were independently replicated two (b, c, e, and g) or three (a, d, f, h, i, and j) times. Starv, starvation; Wort, wortmannin.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Incubation, Stable Transfection, Expressing, Imaging, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: cGAMP induces LC3B lipidation and redistribution of the V1 complex in the perinuclear region that is sensitive to pharmacological inhibition of the V-ATPase. (a and b) WT HeLa cells and primary HDFs were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 for 8 h (a) and 4 h (b). (c) WT HeLa cells and WT HeLa cells with stable expression of GFP-STING were incubated with 15 µg/ml (WT) or 60 µg/ml (WT GFP-STING) cGAMP alone and with 100 nM BafA1 for 8 h. All the following experiments were treated the same unless specified. (d) HDFs were incubated with 60 µg/ml cGAMP alone and with BafA1 for 4 h. (e) FIP200 KO HeLa cells were incubated with cGAMP alone and with BafA1. (f) HDFs were incubated with 60 µg/ml cGAMP alone, with BafA1 and 200 nM wortmannin (Wort) alone, and with cGAMP, BafA1, and Wort for 4 h. (g) Representative Airyscan-processed confocal imaging of FIP200 KO HeLa cells with stable expression of RFP-LC3B and GFP-STING treated with cGAMP (60 µg/ml) alone and with BafA1, fixed, and immunostained for endogenous ATP6V1D. Scale bar, 10 µm, 2 µm (inset). Each Western blotting experiment was independently replicated three times. Starv, starvation.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Inhibition, Incubation, Expressing, Imaging, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: STING activation induced LC3B lipidation is mediated by the WD40 domain of ATG16L1. (a) Schematic representation of domains in the ATG16L1 isoforms (α and β) and ΔWD40 assessed in d. CC, coiled-coil. WIPI2 and FIP200 indicate interacting regions. (b) WT and ATG16L1 KO HeLa cells stably expressing GFP-STING and BFP-ATG16L1-α, BFP-ATG16L1-β, or ATG16L1-ΔWD40 were treated with 60 µg/ml cGAMP for 8 h. (c) Representative Airyscan-processed confocal imaging of FIP200 KO HeLa cells with stable expression of RFP-LC3B and GFP-STING treated with cGAMP (60 µg/ml) alone and with 100 nM BafA1 for 8 h, fixed, and immunostained for endogenous ATG16L1. Scale bar, 10 µm, 2 µm (inset). Each Western blotting experiment was independently replicated three times.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Activation Assay, Stable Transfection, Expressing, Imaging, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: Experiments supplementary to Figs. 3 and ​and55. (a) WT and ATG16L KO HeLa cells with stable expression of GFP-STING and BFP-ATG16L-α, BFP-ATG16L-β, or ATG16L-ΔWD40 were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 for 8 h. All the following experiments use the same conditions unless specified. (b and c) WT HeLa cells and WT HeLa cells with stable expression of BFP-SopF were incubated in starvation media alone and with BafA1 for 4 h (b) and treated with 15 µg/ml cGAMP for 8 h (c). Western blotting experiments were independently replicated three times. (d and e) Representative Airyscan confocal imaging of primary HDFs and HDFs with stable expression of BFP-SopF were treated with 60 μg/ml cGAMP for 4 h, fixed, and immunostained for endogenous ATP6V1D (d) or LC3 (e). Scale bar, 10 µm.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Expressing, Incubation, Western Blot, Imaging

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: The V-ATPase targeting bacterial effector, SopF, blocks STING-mediated LC3B lipidation and perinuclear foci formation. (a) FIP200 KO HeLa cells and FIP200 KO HeLa cells with stable expression of mammalian codon optimized BFP-SopF were incubated with 15 µg/ml cGAMP for 8 h. (b) Representative Airyscan confocal imaging of WT HeLa cells with stable expression of GFP-STING alone and with GFP-STING and BFP-SopF were treated with 60 µg/ml cGAMP for 8 h, fixed, and immunostained for endogenous LC3. Scale bar, 10 µm. (c) WT HeLa cells with stable expression of GFP-STING alone and with either stable expression of BFP-SopF or BFP-SopF-Y224D were incubated with 60 µg/ml cGAMP for 8 h. (d) WT HeLa cells and WT HeLa cells with stable expression of BFP-SopF were incubated with 100 μM monensin for 1 h. Western blotting experiments were independently replicated three times. (e) Model of VAIL onto single-membrane perinuclear vesicles induced by cGAMP activation of STING created with BioRender.com. (1) cGAMP-activated STING translocates from the ER to the Golgi apparatus to colocalize at or around single-membrane perinuclear vesicles. (2) The V1 complex docks to resident V0 domains in perinuclear vesicles, or vesicles with assembled V-ATPases redistribute to a denser formation in the perinuclear region. This process is blocked by BafA1. (3) ATG16L1 is recruited to interact with the V-ATPase via its WD40 domain (as reported by Xu et al., 2019). (4) LC3B is conjugated to phosphatidylethanolamine on single-membrane perinuclear vesicles by the ATG16L1-ATG5-12 complex. This process is inhibited by BafA1 and SopF. Mon, monensin.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Expressing, Incubation, Imaging, Western Blot, Membrane, Activation Assay

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: GFP-LC3B is targeted to perinuclear single-membrane vesicles upon STING activation by cGAMP. (a) Confocal image data (deconvolved; maximum projected) acquired from WT HeLa cells expressing GFP-LC3B (green) and mCh-STING (red), and stained with Hoechst 33342 (blue) after 8 h cGAMP (60 µg/ml) incubation, before correlative imaging of the indicated region of interest (white inset frame). (b–d) Spatial alignment between the (b) optical and (c) electron micrographs acquired within the region indicated in a, (d) with direct overlay between imaging modalities. (e–g) Correlative micrographs of GFP-LC3B–positive structures detected from d. (h–j) Representative electron micrographs of starvation induced autophagosomal structures in WT HeLa cells after 8 h incubation with Earle's Balanced Salt Solution in the presence of BafA1 (100 nM). (k) Confocal image data (deconvolved; maximum projected) acquired from FIP200 KO HeLa cells expressing GFP-LC3B and mCh-STING, stained with Hoechst 33342 after 8 h cGAMP (60 µg/ml) incubation. (l–n) Spatial alignment between (l) optical and (m) electron micrographs acquired at region indicated in k, (n) displayed with direct overlay between micrographs. (o–q) Selected examples of GFP-LC3B–positive structures detected from n. Portions of the Golgi apparatus indicated by asterisk. Scale bars, a–d and k–n, 5 µm; e–j and o–q, 200 nm. EBSS, Earle's Balanced Salt Solution.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Membrane, Activation Assay, Expressing, Staining, Incubation, Imaging

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: VAIL is induced by transfection of poly(dA:dT) independently of STING. (a) WT and FIP200 KO HeLa cells were transfected with 4 μg/ml poly(dA:dT) (dsDNA) and Lipofectamine 2000 at (1:1.5 μg DNA: μl Lipofectamine) alone and with BafA1 (100 nM) for 4 h. All the following experiments were treated the same unless specified. (b) WT and ATG16L KO HeLa cells with stable expression of GFP-STING and BFP-ATG16L-α, BFP-ATG16L-β, or ATG16L-ΔWD40 were transfected with dsDNA. (c) WT HeLa cells and WT HeLa cells with stable expression of BFP-SopF were transfected with dsDNA. (d) WT, STING KO, and STING KO HeLa cells stably expressing GFP-STING were transfected with dsDNA.

Article Snippet: For TEM imaging of starvation-induced autophagosomes , WT HeLa cells were cultured in a six-well plate for 48 h before incubation with Earle's Balanced Salt Solution starvation medium in the presence of 100 nM BafA1 (Bioaustralis) and fixed in prewarmed phosphate-buffered 4% PFA (1 h at 37°C).

Techniques: Transfection, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

doi: 10.1101/2024.12.30.630740

Figure Lengend Snippet: A. U2OS cells were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 72 h. Transfection efficiency was analysed by qPCR where the relative mRNA expression of SYTL5 was normalised to the expression levels of TBP and siControl. Error bars represent the standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Dunnett multiple comparison test, *** = p>0.001. B. U2OS cells expressing the mitochondrial matrix reporter NIPSNAP1 1–53 -EGFP-mCherry (referred to as IMLS cells) were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 h before 24 h incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 h. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the control siRNA and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. C. U2OS IMLS cells expressing PARKIN were transfected with 20 nM control siRNA or SYTL5 siRNA oligos for 48 h before a 16 h incubation with 20 μM CCCP to induce mitophagy, with treatment with 100 nM BafA1 or not for the last 2 h. The area of red-only puncta per cell (>1000 cells analysed) was quantified using Cell Profiler from widefield images obtained using a high-content imaging microscope. The obtained results were normalised to the control siRNA and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Tukeýs multiple comparison test. D. U2OS IMLS cells were transfected with 20 nM control siRNA or RAB27A siRNA oligos for 48 h before 24 h incubation with 1 mM DFP in the absence or presence of 100 nM BafA1 for the last 2 h. After fixation, cell nuclei were stained with Hoechst and widefield images were obtained using a high-content imaging microscope. The area of red-only puncta per cell (>1000 cells analysed) was quantified using a Cell Profiler pipeline. The obtained results were normalised to the siControl, and error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by two-way ANOVA followed by Bonferroni multiple comparison test. E. WT (control) or SYTL5 KO U2OS cells were starved in EBSS for 4 h or incubated with complete medium in the presence or absence of 100 nM BafA1 the last 2 h. Cell lysates were harvested and analysed by western blot for LC3B and actin proteins. LC3B-II band intensity was quantified relative to actin and normalised to control. Error bars represent the mean with standard deviation between replicates (n=3). Significance was determined by one-way ANOVA followed by Bonferroni multiple comparison test.

Article Snippet: Control wells with 100 nM BafA1 (AH diagnostics #BML-CM110-0100) were dosed 2 h prior to fixation.

Techniques: Transfection, Control, Expressing, Standard Deviation, Comparison, Incubation, Staining, Imaging, Microscopy, Western Blot

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of LC3 family protein expression and lipidation. ( A ) Total cell lysates from primary human fibroblasts treated with BafA1, Ex527 or TSA for 48h were analyzed by Western blot as indicated. ( B – E ) Densitometric quantifications of the immunoreactive bands from n = 3 independent experiments. Raw numbers were first normalized to tubulin, and the resulting values were related to the similarly tubulin-normalized control (Ctrl). ( B ) Autophagic flux analyses in cells treated as indicated for 48h. Analysis was performed for each ATG8 protein by determining the difference, between BafA1-treated and BafA1-untreated cells, of the intensity of the lipidated bands running below the unlipidated bands [Flux = (LC3II/Tub) with BafA1 —(LC3II/Tub) without BafA1 ]. Significant changes (by one-way ANOVA on ranks) versus the control: * p ≤ 0.05; ** p ≤ 0.01. ( C ) Raw intensities of all lipidated protein bands (tubulin-normalized). Significant changes (by two-way ANOVA) versus BafA1-untreated cells: * p ≤ 0.05; ** p ≤ 0.01. ( D ) Raw intensities of all lipidated and unlipidated protein bands (tubulin-normalized). Statistical analysis was done as in ( C ). ( E ) Quantification of SIRT1 and p62 expression (tubulin-normalized).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A and LC3B in response to pharmacological sirtuin inhibition. Primary human fibroblasts treated with BafA1, Ex527 or TSA as indicated were immunostained with LC3A, LC3B and p62 antibodies and analyzed microscopically. ( A ) Cellular localization of LC3A, LC3B and p62 after treatment with BafA1 alone. ( B ) Cellular localization of LC3A, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Cellular localization of LC3A, LC3B and p62 after treatment with TSA and BafA1. ( D ) Image analytical quantification of the relative nuclear fraction of LC3A and LC3B in cells treated as indicated and counterstained with DAPI. Significant changes (by two-way ANOVA) versus the control: ## p ≤ 0.01; ### p ≤ 0.001; significant changes between the LC3s: *** p ≤ 0.001. ( E ) Relative co-localization of LC3A and LC3B with p62 quantified by image analysis. Statistical analysis was done as in D. Data in ( D ) and ( E ) were derived from three photographed, independent experiments ( n = 3), in which 10–50 cells per image were analyzed. ( F ) Magnified images of cells immunostained for LC3A or LC3B including the chromatin counterstain (DAPI).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Control, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3C and LC3B in response to pharmacological sirtuin inhibition. Primary human fibroblasts were treated with BafA1, Ex527 or TSA and analyzed as in . ( A ) Cellular localization of LC3C, LC3B and p62 after treatment with BafA1 alone. ( B ) Cellular localization of LC3C, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Cellular localization of LC3C, LC3B and p62 after treatment with TSA and BafA1. ( D ) Quantification of the relative nuclear fraction of LC3C and LC3B in cells treated as indicated. Significant changes (by two-way ANOVA) versus the control: ### p ≤ 0.001; significant changes between the LC3s: ** p ≤ 0.01; *** p ≤ 0.001. ( E ) Relative co-localization of LC3C and LC3B with p62 quantified by image analysis. Statistical analysis was done as in D. Data in ( D ) and ( E ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed. ( F ) Magnified images of cells immunostained for LC3C or LC3B including the chromatin counterstain (DAPI).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Control, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B and LC3C in response to siRNA-mediated Sirtuin1 knock-down. ( A ) Cellular localization of LC3A, LC3B and p62 after transfection with Sirtuin1 siRNA (siSIRT1-RNA) or scrambled RNA (scrSIRT1-RNA) under BafA1 treatment. ( B ) Cellular localization of LC3C, LC3B and p62 after transfection with Sirtuin1 siRNA (siSIRT1-RNA) or scrambled RNA (scrSIRT1-RNA) under BafA1 treatment. ( C ) Quantification of the relative nuclear fraction of LC3A, LC3B and LC3C in cells transfected as indicated. ( D ) Relative co-localization of LC3A, LC3B and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrSIRT1-treated cells: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Data in ( C ) and ( D ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed. ( E ) Sirtuin1 protein expression after transfection with scrSIRT1-RNA or siSIRT1-RNA. Tubulin was used as loading control.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Derivative Assay, Expressing, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B and LC3C in response to pharmacological sirtuin inhibition under starvation conditions. Primary human fibroblasts were grown under starvation conditions and treated with BafA1 and Ex527. ( A ) Cellular localization of LC3A, LC3B and p62 after treatment with Ex527 and BafA1. ( B ) Cellular localization of LC3C, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Quantification of the relative nuclear fraction of LC3A, LC3B and LC3C in cells treated as indicated. ( D ) Relative co-localization of LC3C, LC3B and LC3A with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus starvation-only treated cells: ** p ≤ 0.01; *** p ≤ 0.001. Data in ( C ) and ( D ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Western blot analysis of the cellular localization of LC3A, LC3B and LC3C in response to pharmacological sirtuin inhibition under normal and starvation conditions. Primary human fibroblasts were grown under normal or starvation conditions and were treated with BafA1, Ex527 or TSA, as indicated. Nuclear and cytosolic fractions of the cells were prepared and investigated for their protein content of LC3A, LC3B and LC3C. The nuclear marker histone H3 and the cytosolic marker tubulin were analyzed in selected fractions for general separation quality control purposes.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Western Blot, Inhibition, Marker, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3B knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3B-RNA, ( B ) siLC3B-RNA, ( C ) siLC3B-RNA and Ex527 treatment, ( D ) siLC3B-RNA and starvation, and ( E ) siLC3B-RNA and starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3A and LC3C in cells treated as indicated. ( G ) Relative co-localization of LC3A and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3B: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3B: # p ≤ 0.05; ## p ≤ 0.01. ( H ) LC3B protein expression in cells transfected with scrLC3B-RNA or siLC3B-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3A knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3A-RNA and ( B ) siLC3A-RNA, (C) siLC3A-RNA and Ex527 treatment, ( D ) siLC3A-RNA and starvation, and ( E ) siLC3A-RNA and starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3B and LC3C in cells treated as indicated. ( G ) Relative co-localization of LC3B and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3A: ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3A: # p ≤ 0.05; ## p ≤ 0.01. ( H ) LC3A protein expression in cells transfected with scrLC3A-RNA or siLC3A-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3C knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3C-RNA, ( B ) siLC3C-RNA, ( C ) siLC3C-RNA and Ex527 treatment, ( D ) siLC3C-RNA and starvation, and ( E ) siLC3C-RNA, starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3A and LC3B in cells treated as indicated. ( G ) Relative co-localization of LC3A and LC3B with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3C: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3C: # p ≤ 0.05; ### p ≤ 0.001. ( H ) LC3C protein expression in cells transfected with scrLC3C-RNA or siLC3C-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three images per treatment from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay