EMBO/soda-nel huggingface dataset:
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 6

Figure 6. Laminin 511 stabilizes VE-cadherin at cell-cell junctions.

A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test. C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511. D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test. E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.
Endothelial Basement Membrane Laminin 511 is Essential for Shear Stress Response (2016)
Figure 7

Figure 7. Laminin 511 affects the tension of endothelial cells adherens junctions in vivo.

A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf1

(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS–PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI–TOF MS. HC, heavy chain. (b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2–77, 2–275 and 232–607) of Flag–Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12–Atg5. Total lysates were run alongside as controls for protein input. FL, full-length. (c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2. (d) HeLa cells transfected for 24 h with Flag–Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin–Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf2

(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/− BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin. (b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001. (c) HeLa cells stably expressing GFP–mRFP–LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells. (d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf3

(a–d) HeLa cells transfected for 72 h with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA were treated with HBSS (to induce autophagy) for 6 h, after which they were fixed and immunostained for endogenous Atg16L1. Representative cells for the control and epsin 1 siRNA are in a, and percentages of HeLa cells with Atg16L1 vesicles are quantified in b (for epsin 1), c (for clathrin) and d (for AP1 and AP2). Three asterisks, P 0.0001. Scale bars, 10 μm. (Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain). n = 600 cells. (e, f) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2–275 Atg16L1 or 232–607 Atg16L1 were fixed and immunostained with anti-Flag antibody (e); the proportions of cells with Flag–Atg16L1 vesicles are represented in f. Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e. The large boxes in each panel are higher magnifications of regions in the smaller boxes. Three asterisks, P 0.0001; n.s., not significant. n = 100 cells. (g–i) HeLa cells transfected with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA, as above, were transfected with GFP–Atg16L1 and tomato LC3 for a further 24 h, after which the cells were fixed. g shows representative cells for control, epsin 1 and clathrin siRNA. The percentage of GFP–Atg16L1 (green) that co-localized (yellow, marked by arrows) with tomato LC3 vesicles (red) is quantified as shown in the graph in h (control, epsin 1 and clathrin siRNA) and i (control, AP2 and AP1 siRNA). Three asterisks, P 0.0001. n = 21 cells. Scale bars, 10 μm. All error bars represent s.e.m.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf4

(a) HeLa cells transfected for 24 h with GFP–Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm. (b) HeLa cells transiently transfected for 20 h with GFP–Atg16L1 and mRFP–GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP–Atg16L1 (green) and mRFP–GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP–Atg16L1 with mRFP–GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm. (c) HeLa cells transfected for 24 h with GFP–Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf5

(a) HeLa cells transfected for 24 h with GFP–Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm. (b) HeLa cells transfected for 24 h with GFP–Atg16L1 (green) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) as in a, after which they were fixed, immunostained for endogenous (end.) EEA1 (blue) and analysed by confocal microscopy. Vesicles positive for Atg16L1 and cholera toxin B are yellow (white arrows) and vesicles positive for both EEA1 and cholera toxin are purple (blue arrows) and are shown in the high-magnification images in the middle. The graph shows percentage co-localization. Cho. tox., cholera toxin. n = 20 cells. Scale bar, 10 μm. (c) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed, and immunostained for endogenous Atg16L1 (green) and endogenous EEA1 (blue). Cells were analysed as in b; the graph shows quantification of the results. Scale bar, 10 μm. Arrows indicate Atg16L1–cholera toxin co-localization; arrowheads indicate EEA1–cholera toxin co-localization. n = 20 cells. (d) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed and immunostained for endogenous Atg5 (green) or endogenous Atg12 (green). Co-localization of the Atg5 (n = 18 cells) or Atg12 vesicles (n = 39 cells) with cholera toxin is shown in yellow (arrows) and is quantified in the graph. Scale bar, 10 μm. All error bars represent s.e.m.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf6

(a) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2–275 or 232–607 Atg16L1, together with GFP–Atg5 and CFP–LC3, were fixed and immunostained with anti-Flag antibody before confocal analysis. Co-localization of wild-type or 2–275 Atg16L1 (red) with GFP–Atg5 (green) is marked by arrows in the insets. Scale bars, 10 μm. (b) HeLa cells transfected for 24 h with GFP–Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
figf7

(a) HeLa cells transfected for 24 h with GFP–Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP–Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP–Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels. (b, c) HeLa cells transfected with control, clathrin heavy chain, AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP–Atg16L1 (green) together with the siRNA for the next 24 h. The cells were then incubated for 15 min with Alexa Fluor-555-conjugated cholera toxin subunit B (red) at 4 °C, followed by further incubation for 10 min at 37 °C, after which the cells were fixed for confocal analysis (b). The co-localization (yellow) of GFP–Atg16L1 (green) with cholera toxin (red) in control or clathrin heavy-chain knockdown is shown and is quantified in the graphs in c. n = 25 cells. Three asterisks, P = 0.0002 for clathrin heavy-chain knockdown, and P 0.0001 for AP2 knockdown. All error bars represent s.e.m. Scale bar, 5 μm.
Plasma membrane contributes to the formation of pre-autophagosomal structures (2010)
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(a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot for total lysate (bottom) and IP (top) were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1–clathrin interaction with AP2 knockdown. The graph shows quantification of the Atg16L1–clathrin or Atg16L1–AP2 with knockdown of control, clathrin heavy chain or AP2 from two independent experiments. (b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP–PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole. (c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1–clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m. d, Representation of the contribution of the plasma membrane to autophagosome precursor formation. Cholera toxin is internalized by clathrin-dependent and clathrin-independent endocytosis. Clathrin knockdown significantly decreases cholera toxin uptake. Clathrin-coated vesicles (CCV) budding immediately from the plasma membrane (EEA1-negative) are precursors to early endosomes (EE) (EEA1-positive). Previous studies showed that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes or multivesicular bodies (LE/MVB) to form amphisomes, which are Atg16L1-negative, LC3-positive and positive for endosomal markers (red arrows). We show here that inhibition of clathrin-dependent internalization inhibits the formation of early Atg16L1-positive precursors that mature to form phagophores and, later, autophagosomes (blue arrows). These Atg16L1 vesicles were positive for other early autophagosomal markers (Atg5 and Atg12) but not for early endosomal markers (EEA1). AP2 is a clathrin adaptor at the plasma membrane, whereas AP1 localizes to the trans-Golgi network and endosomes. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease (2021)
Figure 1

Figure 1. Fluc-EGFP reacts to proteostasis changes in primary neurons.

A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes.

B, Quantification of FlucSM-EGFP foci formation in transfected neurons. N=4 biological replicates for BafA1 and corresponding control group, 3 biological replicates for all the other conditions; Two-tailed t-test.

C, Left, quantification of specific luciferase activity of FlucSM-EGFP upon indicated treatments, normalized to respective vehicle-treated controls. N=3 biological replicates; One-sample t-test. Right, representative Western blot of neuronal lysates for the indicated conditions. Tubulin was used as a loading control.

D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D.

E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph.

Data information: Error bars represent SD. Significance: *p<0.05, **p<0.01, ***p<0.001. Scale bars: A and D, 5 µm; insets in A, 2 µm.
Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease (2021)
Figure 4

Figure 4. Fluc-EGFP reporter does not detect proteostasis impairment in R6/2 mice.

A, Cortical (upper row) and striatal (lower row) sections from 12-week-old R6/2:Fluc-EGFP mice (right) and control WT:Fluc-EGFP littermates (left) stained for aggregated mHTT (EM48, magenta). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were counterstained with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes. Continuous and stippled lines mark cell bodies and nuclei, respectively.

B, C, Luciferase assay in R6/2 mice. B, Left, scheme of brain regions used for luciferase assay color-coded by their vulnerability to Huntington's disease. Right, specific luciferase activity measured in the indicated brain regions of 12-week-old R6/2:Fluc-EGFP mice normalized to WT:Fluc-EGFP littermates. C, Fluc-EGFP protein quantity in the indicated brain regions of R6/2:Fluc-EGFP mice measured by Western blot. Values are normalized to WT:Fluc-EGFP littermates. N=5 mice of each genotype. Colored asterisks in B and C indicate significant comparisons to the respective brain regions of control littermates (one-sample t-test).

Data information: Error bars represent SD. Significance: *p<0.05; **p<0.01. Scale bars in A, 10 µm.
Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease (2021)
Figure 5

Figure 5. Response of nuclear and cytoplasmic Fluc-EGFP to nuclear and cytoplasmic mHTT. A, B, Primary cortical neurons transfected with cyt-Fluc-EGFP (A) or nuc-Fluc-EGFP (B) (green), in combination with nuc-mHTT (upper rows) or cyt-mHTT (lower rows) (magenta). Cells were fixed at DIV 3+2 and stained for the neuronal marker MAP2 (grey). Nuclei were labeled with DAPI (blue). Arrowheads point to Fluc-EGFP foci. Dashed lines mark the nuclei. Schemes on the right summarize the cellular distribution of the respective Fluc-EGFP (green) and mHTT (magenta) proteins. Corresponding cultures transfected with control HTT are shown in Appendix Fig. S3A.

C, Quantification of Fluc-EGFP foci formation. N=3 independent experiments. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, ****p<0.0001; Fluc, n.s.; Interaction HTT x Fluc, n.s. Significant pairwise comparisons within the respective Fluc-EGFP group are indicated above the bars.

D, Quantification of the subcellular localization of the respective Fluc-EGFP protein in the presence of the indicated mHTT constructs. N=3 independent experiments. Three-way ANOVA with Tukey's multiple comparisons test. ANOVA: Localization, *p=0.019; HTT, n.s.; Fluc, n.s.; Localization x HTT, **p=0.0012; Localization x Fluc, ****p<0.0001; HTT x Fluc, n.s.; Localization x HTT x Fluc, ***p=0.0009.

E, Quantification of Fluc-EGFP foci localization in the presence of cyt-mHTT. N=3 independent experiments. Two-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Localization, ****p<0.0001; Fluc, n.s.; Localization x Fluc, *p=0.01. Dark-green, light-green and striped asterisks in D and E indicate significant comparisons of the fractions of cells with Fluc-EGFP localized in the cytoplasm, in the nucleus, or in both compartments, respectively.

Data information: Error bars represent SD. Significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Scale bar in A, B, 5 µm.

Expanded View figure legends
Synthetic lethal metabolic targeting of cellular senescence in cancer therapy (2013)
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a, BrdU incorporation reflecting DNA synthesis and SA-&bgr;-gal activity as markers of CTX-induced senescence in Eµ-myc transgenic control;Bcl2 versus Suv39h1−;Bcl2 lymphomas in vivo. Numbers represent mean percentages of positive cells ± s.d. (n = 5 each). b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups. c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1−;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d–g). Results represent means ± s.d. (n = 5 each). d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas). e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro (n = 3 each; &agr;-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each). g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P  0.05.