EMBO/soda-nel huggingface dataset:
Macrophage-driven nutrient delivery to phagosomal S. aureus supports bacterial growth (2020)
Figure 3

Figure 3. The uptake of macromolecules from the extracellular environment can be inhibited pharmacologically.

(A) Uptake of Alexa® Fluor647-conjugated transferrin by RAW 264.7 macrophages in the presence and absence of PIK-III and Dynasore. The fluorescence micrographs show transferrin (in blue) and the macrophage plasmalemma demarcated by TMR-conjugated wheat germ agglutinin (in red). The dashed line indicates the region of the cell for which the line scan analysis, shown graphically on the right, was done. These micrographs are representative images obtained from three independent experiments. Line scan analysis was performed to indicate the distribution of fluorescent transferrin (blue line) relative to the plasmalemma (red line) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs. Scale bars equal 10 μm.

(B) RAW macrophages were pre-treated with vehicle (DMSO) or the vps34 inhibitor PIK-III prior to the addition of 10 kDa FITC-dextran (100 µg/mL) for 2 hrs. Prior to imaging live, macrophages were also stained with Cy5-conjugated WGA. The representative micrographs depict inhibited dextran uptake indicating PIK-III treatment can perturb fluid phase uptake. Scale bars equal 10 µm.

(C) The intensity of dextran fluorescence was quantified for RAW cells pre-treated with vehicle, PIK-III (10 µM), Dynasore (100 µM) or PIK-III and Dynasore (10 and 100 µM) respectively. The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments. * indicates p<0.05, as determined by an ordinary one-way ANOVA with a Bonferroni multiple comparisons test.
Macrophage-driven nutrient delivery to phagosomal S. aureus supports bacterial growth (2020)
Figure 5

Figure 5. Inosine monophosphate dependent growth of a purK S. aureus in macrophages requires macrophage-driven fluid-phase uptake.

(A) Growth of wild-type S. aureus USA300 or a purK mutant. The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI. Additives are indicated on the x axis and were either 5% (v/v) FBS, 50 μM IMP, 10 μM PIK-III and 100 μM Dynasore or an equivalent volume of DMSO. Each symbol represents a biological replicate of S. aureus and the data derive from three independent experiments. Statistical significance was determined by a one-way ANOVA with a Holm-Sidak's multiple comparison test. n.s. indicates not significant, *** indicates p<0.0001.

(B) The graph depicts the fold change in bacterial burden for RAW 264.7 macrophages infected with purK S. aureus. Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK-III and Dynasore which were maintained throughout the infection. The data are the mean ± standard deviation of the burden at 10 h post-infection normalized to the burden at 1.5h post-infection prior to the addition of IMP. Each symbol represents a single biological replicate of S. aureus and derive from three independent experiments. Significance was determined by one-way ANOVA with a Turkeys multiple comparison test. *p<0.05, **p<0.01.

(C, D) The ability of purK S. aureus to grow in an IMP-dependent manner within primary bone marrow-derived macrophages is shown. (C) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non-replicating bacteria are both GFP and proliferation dye positive. In contrast, replicating bacteria (white arrows) are only GFP positive. The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S. aureus in the presence of 50 μM IMP. Images were acquired at 12 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (D) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs. Each data point represents a biological replicate (n=8) and derived from three independent experiments. The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition. ** indicates p<0.01 as determined by an unpaired two-tailed t-test with a Wilcoxon matched pairs test.
Loss of all three APP family members during development impairs synaptic function and
plasticity, disrupts learning and causes an autism-like phenotype. (2021)
Figure 1

Fig 1: NexCre cTKOs show agenesis of the corpus callosum and abnormal hippocampal lamination

A: Stereological evaluation of cortical volume of young (Y-WT, Y-LM, Y-cTKO, age: 5-6 months, each group n=5) and old (O-LM n=5, O-cTKO n=6, age: 18-20 months) mice in 12 equidistant bins along the caudal-rostral axis of the coronally sectioned brain. Each bin would comprise an approximately 570 µm thick slab of the brain. No significant differences between genotypes or age groups were seen in the volumes per bin or in the volume distributions along the caudal-rostral axis. Areas shaded in grey correspond to the 95% confidence intervals.

B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm.

C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm).

D: Quantification of the number of CA1 cells in WT (white, n=5, age: 5-6 months), LM (grey, n=5, age: 18-20 months) and NexCre cTKO (red, n=6, age: 18-20 months) mice. One-way ANOVA revealed no significant difference between genotypes (p = 0.0814). Data are represented as mean + SD.

E: Quantification of callosal malformations. n = 10 LM and n = 11 NexCre cTKO animals.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf1

(a) A schematic diagram of the ubiquitin-like lipidation reaction of autophagy. (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8–12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS–PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7–GRL1 conjugate, the Atg3–GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein. (c) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC. (d) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS–PAGE and imaged by Coomassie. For c and d, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the vertical lines. Uncropped versions of all gels are included in Supplementary Fig. 7.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf2

(a) A schematic diagram depicting incompatibility of idealized cone-shaped lipids with planar assemblies. The spontaneous curvature in a lipid monolayer scales qualitatively with the idealized shape of the lipid where the idealized volume can be approximated as a cylinder (PC) or a cone (PE). When membrane curvature deviates significantly from the molecular spontaneous radius of curvature for an individual lipid, packing defects arise forcing lipids to adopt sub-optimal configurations, producing local monolayer stress. Resolution of the local stress associated with these incompatibilities can include changes in lipid structure, a reorganization of lipid composition or the insertion of non-lipidic molecules including proteins. (b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05. (c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf3

(a) Membrane defects resembling those accumulating with high concentrations of PE are prevalent in vivo at sites of high curvature and can be recapitulated in vitro by forming liposomes of different diameters. (b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of PE observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of DOPE (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL1, GL2 and LC3 proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the PE is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient. (c) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf4

(a) The accumulation of the Atg3–GL1 conjugate depends on the extent of membrane curvature. In vitro GL1 coupling reactions were run as in Fig. 1. The reactions were visualized by immunoblot against Atg3 (top) and by Coomassie staining of GRL1 (bottom). The accumulation of Atg3–GL1 is inversely related to the formation of the lipidated GRL1, suggesting that the membrane-curvature-dependent step may involve Atg3 recognition of the membrane surface. (b) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30 °C for 90 min with liposomes containing 0, 30 or 55 mol% DOPE (5 mM lipids) and of varying sizes (extrusion membrane dimensions are shown on the figure; actual final sizes were determined by dynamic light scattering (Supplementary Table 1)). The liposome-associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS–PAGE. (c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf5

(a) Helical wheel representation of the predicted N-terminal amphipathic helix (amino acids 4–26) of mouse Atg3 (wheel generated in Heliquest). (b) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400 nm and sonicated liposomes (30 mol% DOPE) with the indicated Atg3 mutants. The extent of GL1–PE formation (top gel) was determined from densitometry and is plotted as percentage of total GL1 (bar graph). The ability to form the Atg3–GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. In both panels, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the lines. (c) Curvature dependence of K11L mutant binding to liposomes compared with wild-type Atg3. The binding reaction was performed as in Fig. 4b on 30 DOPE% liposomes. F, float-up; T, total input. (d) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in b. Total GL1 lipidation was assessed from reactions with wild-type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. Left: Coomassie gels of individual lipidation reactions. Right: densitometry of Coomassie-stained gels revealed the ratio of GL1–PE to the sum of all three GL1 species as in Fig. 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. For b,c, P values were calculated between sonicated and 400 nm samples of the same mutant. For d, P values represent comparison with WT protein on the same liposome size and composition. For all panels, n = 3 independent experiments, error bars represent standard deviation and **P 0.05; *P 0.01. Uncropped gels in Supplementary Fig. 7.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 (2014)
figf6

Atg3-knockout MEFs (Atg3−/−) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To determine whether the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3−/− MEFs by lentiviral infection and rescue of the three phenotypes was tested. (a) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a nonspecific band recognized by the Atg3 antibody. (b) Cells were infected as in a and transduced cells were selected with puromycin for 1–2 days before starvation and immunolabelling with the anti-LC3B antibody. Representative images are shown in b. (c) The number of LC3 puncta per cell was counted manually from 22–96 cells per replicate. Raw data can be found in Supplementary Fig. 5. P values represent difference from vector negative control. n = 2–5 independent experiments; the precise n values and number of cells per experiment are listed in the Methods. (d) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti-Atg16L antibody. Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods. This ratio was plotted as arbitrary units. Grey dashed line indicates the value for Atg3−/− cells rescued with wild-type Atg3. n = 2 (for V15K) or 3 (for all others) independent experiments; the precise n values and number of fields visualized per experiment are listed in the Methods. Raw data can be found in Supplementary Fig. 4. Atg3 variants that support lipidation of LC3 family proteins in vitro (Fig. 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. P values represent difference from wild-type rescue. **P 0.1; *P 0.05. Error bars in c,d represent s.d. for all samples of n > 2. Uncropped versions of all gels are included in Supplementary Fig. 7.
Autophagosomes form at ER–mitochondria contact sites (2013)
f1

a, COS7 cells co-transfected with GFP–ATG14 and DsRed–ER or RFP–SEC61&bgr; (an ER marker) were immunostained for the mitochondria marker TOMM20. Scale bars, 2 &mgr;m. b, An immunogold electron micrograph of starved HeLa cells expressing GFP–ATG14 probed with anti-GFP antibodies (red arrows). Scale bar, 200 nm. c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP–DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.
Autophagosomes form at ER–mitochondria contact sites (2013)
f2

a, COS7 cells stably expressing yellow fluorescent protein (YFP)–ATG5 were transfected with RFP–SEC61&bgr; and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20. b, Time-lapse images of COS7 cells transfected with YFP–ATG5, RFP–SEC61&bgr; and TFP-mito under starved conditions. c, d, Distribution of distances of YFP–ATG5 dots from the centre to the surface of the ER (c) and to the surface of mitochondria (d). e, f, Distances of YFP–ATG5 dots over their lifetime for data from c (e) and d (f), respectively. All scale bars, 2 &mgr;m.
Autophagosomes form at ER–mitochondria contact sites (2013)
f3

a, COS7 cells expressing GFP–SEC61&bgr; and STX17 were immunostained for TOMM20. b, HeLa cells expressing GFP–ATG14 were immunostained for STX17. c, Immunoblot of subcellular fractions of HEK293 cells. d, STX17 knockdown COS7 cells expressing GFP–ATG14 and RFP–SEC61&bgr; were immunostained for TOMM20. Error bars denote s.d. e, Immuoblot of subcellular fractions of STX17 knockdown (using two STX17 siRNAs, STX17-1 and STX17-2) HEK293 cells expressing GFP–DFCP1. f, Co-immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag–ATG14 and Myc–STX17. g, Pull-down of ATG14 complex from HeLa cells expressing Myc–STX17 or Myc. Scale bars, 5 &mgr;m (a, b) and 2 &mgr;m (d).
Autophagosomes form at ER–mitochondria contact sites (2013)
f4

a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP–LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole. b, Electron micrographs of STX17 knockdown cells. White arrows denote autolysosomes, black arrows denote autophagosome, white arrowheads mark isolation membranes. c, Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP–LC3. d, GFP–ATG5 dots (arrows) in STX17 knockdown HeLa cells. e, Quantification of staining in d. f, Immunoblot of p62 in STX17 knockdown cells. Tubulin was used as a loading control. g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 &mgr;m (a, b), 200 nm (c) and 5 &mgr;m (d).
HDAC inhibitors tune miRNAs in Extracellular Vesicles of Dystrophic Muscle-Resident Mesenchymal Cells (2020)
Figure 2

Fig. 2: Increased amounts of miR-206-3p in DMD muscles, associate with muscle regeneration and inversely correlate with disease progression.

(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.

(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.

(C) Graph showing the fusion index of MuSCs in the conditions described in (B) (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by Tukey test relative to MuSCs alone (-); *p<0.05, ****p<0.0001. Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-culture with FAPs CTR transfected with scramble (Scr); ##p < 0.01. Cross (†) means Tukey analysis compared to MuSCs in co-culture with FAPs TSA transfected with scramble (Scr): ††††p < 0,0001. § represents statistical analysis by 2way Anova test. §§§p < 0,001.

(D) Table representing a manually assembled list of microRNAs revealed by microarray analysis and statistically induced by TSA in FAPs-derived EVs.

(E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2).

(F) Graph representing miR-206-3p violet area quantification relative to (E).

(G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E).

(H) Graph showing the percentage of eMyHC positive fibers of condition described in (G).

(I) Representative images of Masson's Trichrome staining in conditions described in (E).

(J) Graph showing the fibrotic area quantification of conditions described in (I).

Data information: For E,G, I Scale bar = 50 μm.