EMBO/soda-nel huggingface dataset:
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 5

Increasing VEGF doses inhibit activation of endothelial SMAD2/3

Relative gene expression of Id1, specifically induced by activated SMAD1/5, and Pai1, specifically induced by activated SMAD2/3, in total muscle tissues 1 week after implantation with V Low-, V Med- and V High-expressing myoblast clones or control cells (Ctrl), expressed as fold-change versus the Ctrl group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low versus Ctrl P = 0.0002; V Low versus V Med P = 0.005; V Low versus V High P = 0.0016.

Immunofluorescence staining of endothelium (CD31, in red), nuclei (DAPI, in blue), and phosphorylated SMAD2/3 (in cyan) on cryosections of similarly injected muscles confirmed that endothelial SMAD2/3 activation was inhibited by increasing VEGF doses. Scale bar = 10 μm; n = 3.

Source data are available online for this figure.
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 6

TGF-β1 regulates Sema3A production and NEM recruitment

A–C Endothelial Sema3A expression is directly inhibited by VEGF and stimulated by TGF-β1 in vitro. Mouse aortic endothelial cells were stimulated with increasing doses of recombinant VEGF164 or TGF-β1 for 24 h. The expression of Sema3a and Tgfb1 was quantified by qRT-PCR and expressed as fold-change versus the non-stimulated cells. VEGF dose dependently inhibited Sema3a expression (A), but did not affect that of Tgfb1 (B), while TGF-β1 upregulated Sema3a expression in a dose-dependent fashion (C). Data represent individual values, with mean ± SEM (n = 4); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A expression upon VEGF stimulation: 40 versus 0 P = 0.0041; TGF-β1 upon VEGF stimulation: no significant differences; Sema3A expression upon TGF-β1 stimulation: 1 versus 0 P < 0.0001; 2.5 versus 0 P < 0.0001; 40 versus 0 P < 0.0001.

D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (D) Immunohistochemistry for Sema3A protein on frozen muscle sections with H&E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 50 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 10; Ctrl IgG1, n = 17; Ctrl AbαTGF-β1, n = 16; V Low IgG1, n = 23; V Low AbαTGF-β1, n = 28. **P < 0.01 and ***P < 0.001 by Kruskal–Wallis analysis with Dunn’s multiple comparisons test: V Low IgG1 versus No stain P = 0.0069; V Low IgG1 versus Ctrl IgG1 P < 0.0001; V Low AbαTGF-β1 versus V Low IgG1 P = 0.0002. (E) Quantification of recruited NEM (number of CD11b+ cells/mm2 of area). Scale bar = 25 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG1, n = 20; Ctrl AbαTGF-β1, n = 19; V Low IgG1, n = 20; V Low AbαTGF-β1, n = 27; ***P < 0.001 by Kruskal–Wallis analysis with Dunn’s multiple comparisons test: V Low IgG1 versus Ctrl IgG1 P = 0.0005; V Low AbαTGF-β1 versus V Low IgG1 P < 0.0001.

Source data are available online for this figure.
Structural basis of TRAPPIII-mediated Rab1 activation (2021)
Figure 4

Figure 4: The C-terminus of Trs85 binds to the TRAPP core

Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.

As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.

As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.

Network map showing the relative positions of cross-links between the C-terminus of Trs85 and the core subunits Trs31 and Trs20.

View of the cryo-EM structural model showing the interaction surface between Trs85, Trs31 and Trs20. Inset depicts several basic residues from Trs85 that are in close proximity to several acidic residues of Trs20.

Yeast complementation assays performed in a sensitized strain (BET3-GFP::HIS3, trs85∆::KANMX) to assess the functionality of Trs85 mutants within the region shown in E. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)

Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)

Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)
Structural basis of TRAPPIII-mediated Rab1 activation (2021)
Figure 6

Figure 6: TRAPPIII uses a conserved amphipathic helix to bind the membrane

Refined atomic model of the TRAPPIII complex shown as it likely interacts with membranes. The arrow points to the location of the unmodeled conserved region.

Sequence alignment of the unmodeled region predicted to lie on the membrane-binding surface. Red helix above represents the region predicted to fold as an α-helix in secondary structure analysis. Positively charged residues are highlighted in blue, while bulky aromatic residues are underlined.

Helical wheel representation of the region predicted to be an α-helix. Positively charged residues are colored blue, polar uncharged residues are colored green, and nonpolar residues are colored yellow.

The wild-type (WT) Trs85 loop sequence is shown on top, with targets of substitution colored purple and specific substitution mutants color coded.

Yeast complementation assays performed in a sensitized strain (BET3-GFP::HIS3, trs85∆::KANMX) to assess the functionality of Trs85 substitution mutants. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)

Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 substitution mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)

In vitro membrane pelleting assay examining two Trs85 substitution mutants in the context of the TRAPPIII complex. In the absence of liposomes, all three complexes pellet slightly, however in the presence of membranes, only the wild-type Trs85-TRAPPIII complex is strongly enriched in the pelleted fraction. Representative of n=3 independent experiments. (S=supernatant, P=pellet)

Quantification of G. Average % protein pelleted between three assays is plotted, the error bars represent the 95% confidence intervals. The value for each replicate is depicted. The negative values arise from subtraction of the average amount of protein that pellets in the absence of liposomes. Ordinary one-way ANOVA with Tukey's multiple comparisons test: WT vs. 7A and WT vs. 4E **** p < 0.0001, 7A vs. 4E * p = 0.0222.

Live cell fluorescence microscopy of Trs85-mNeonGreen substitution mutants and mRFPmars-Ypt1. Scale bar = 2 μm. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)

In vitro nucleotide exchange assay of prenylated-Ypt1/GDI substrate in the presence of synthetic liposomes. The wild-type Trs85-TRAPPIII complex (purple) is significantly more active than the substitution mutants (red and black) or the core alone (yellow). Representative of n=3 independent experiments.

Assays performed as in J, but using a non-prenylated Rab1/Ypt1 substrate and in the absence of liposomes. Exchange activity of all complexes are equivalent. Representative of n=3 independent experiments.
Structural basis of TRAPPIII-mediated Rab1 activation (2021)
Figure 7

Figure 7: The membrane-binding helix is critical for autophagy

GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)

Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p = 0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)

Model for TRAPPIII-mediated Rab1 activation in cells. Inactive Rab1-GDP/GDI exists in an equilibrium where Rab1-GDP samples various organellar membranes. TRAPPIII is recruited to specific membranes containing negative charges (red circles) and utilizes an amphipathic helix to bind stably to the membrane surface. Rab1 is locally activated by TRAPPIII in order to recruit downstream effectors to facilitate trafficking or autophagic events.

Expanded View Figure Legends
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf1

C38 and IB3-1 cells transfected with GFP–LC3 plasmid in nutrient-rich or starvation medium. (a) Top: confocal microscopy of GFP. N, nucleus. Scale bar, 10 μm. Bottom: percentage of cells containing more than five GFP–LC3 punctate dots per cell. Means ± s.d. (n = 5). Asterisk, P 0.001 versus C38 cells; analysis of variance (ANOVA). (b) Left: electron microscopy of starved C38 and IB3-1 cells. The right-hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells (arrows) and perinuclear mitochondria in IB3-1 cells. N, nucleus. Scale bar, 250 nm. Right: numbers of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus C38 cells; ANOVA. (c) Fluorescence-activated cell sorting (FACS) analysis after staining with fluorescent dye DiOC6 in starved C38 and IB3-1 cells. (d, e) Immunoblot of LC3 in starved C38 and IB3-1 cells (d) and IB3-1 cells (e) after incubation with E64d and pepstatin A. (f, g) Immunoblots (IB) of polyubiquitin (f) and p62 (g) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (h) Immunoblot of LC3 and p62 in control (n = 10) and CF (n = 10) human nasal mucosa. (i) Number of LC3-positive dots (per 104 mm−2 of mucosa) in control (n = 10) and CF (n = 10) human nasal mucosa. Means ± s.d. Asterisk, P 0.01 versus controls; ANOVA. (j) Confocal microscopy of p62 in control and CF human nasal mucosa. Representative images from ten patients with CF. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 10 μm. (k) Number of LC3-positive dots in lung tissues from wild-type (WT) and CftrF508del mice. Means ± s.d. for lung tissues from ten mice per group. Asterisk, P 0.001 versus WT mice; ANOVA. (l) Confocal microscopy of p62 in WT and CftrF508del homozygous mice. Representative staining from at least five airway epithelial areas per mouse (n = 10 mice per group). L, lumen. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. β-Actin was used as loading control for immunoblot analysis of cells, and αβ-tubulin for nasal mucosa. All expreriments were repeated three times. Densitometric analysis of blots is shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf2

(a–d) Starved C38 and IB3-1 cells. (a–c) Confocal microscopy images of beclin 1 (green) and hVps34 (red) (a), calnexin (green) and hVps34 (red) (b), and beclin 1 (green) and HDAC6 (red) (c, left) or hVps34 (green) and HDAC6 (red) (c, right). N, nucleus. Scale bar, 10 μm. Yellow indicates co-localization. (d) Immunoblot of Ambra1, hVps15, hVps34, Atg14L and beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (e–i) Starved IB3-1 cells transfected with either HA-tagged human beclin 1 or the empty vector. (e) Confocal microscopy images of HA (red) and calnexin (green) (top) or hVps34 (red) and calnexin (green) (bottom). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (f) Left: confocal images of LC3-positive dots. N, nucleus. Scale bar, 10 μm. Right: percentage of cells containing more than five LC3-positive dots. At least 60 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA. (g) Left: electron microscopy of HA–beclin 1-transfected IB3-1 cells shows areas enriched in autophagosomes (arrows). Scale bar, 250 nm. Right: number of autophagosomes per cell; 20 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA. (h) Immunoblot of HA and LC3. (i) Immunoblot of p62. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (j) Immunoblot of beclin 1 in WT and CftrF508del mice. Representative of seven mice for each group. (k, l) CftrF508del mice (n = 3 per group) were administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (k) LC3-positive dots per mm2 of lung tissues. Means ± s.d. for lung tissues from three mice per group. (l) Confocal microscopy micrographs of p62. L, lumen. Representative stainings from at least five airway epithelial areas per mouse. Scale bar, 10 μm. β-Actin was used as loading control for immunoblot analysis of cells, and αβ-tubulin for mouse tissue. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf3

(a) IB3-1 cells cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides. IP, immunoprecipitation; IB, immunoblot. (b) IB3-1 cells cultured with the proteasome inhibitor MG132 (50 μM). (c, d) IB3-1 cells transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (c) Cells immunostained with anti-beclin 1 (green) and anti-HDAC-6 (red) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (d) FRET analysis of beclin 1-Alexa 546 (Al546) fluorescence after HDAC6-Cy5 photobleaching. F/F0 indicates the post-bleaching/pre-bleaching fluorescence ratio of Al546/Cy5. (e) IB3-1 cells were transfected with either human TG2 siRNA or scrambled oligonucleotides. Immunoblot of TG2, Ambra-1, hVps15, hVps34, Atg14L and beclin 1 in IB3-1 cells grown in starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (f) IB3-1 and C38 cells were transfected with either 1 μg of pLPCX-WT-TG or pLPCX-Cys277Ser-TG or the empty vector. Immunoblot of beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM human PIASy siRNA or scrambled oligonucleotides. (g) Percentage of cells containing more than five GFP–LC3 punctate dots. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA. (h) Left: electron micrographs of IB3-1 cells showing areas enriched in autophagosomes (arrows) on PIASy gene silencing, EUK-134 or cystamine. Scale bar, 100 nm. Right: number of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA. (i) IB3-1 cells treated with cystamine. Immunoblot of p62. (j) IB3-1 cells treated with cystamine or EUK-134 or transfected with human PIASy siRNA or scrambled oligonucleotides. Immunoblot of beclin 1 (left) and PIASy (right). Uncropped images of blots are shown in Supplementary Information, Fig. S10. The experiments were repeated three times. β-actin was used as loading control for all blots. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf4

(a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM). (a) Immunoblot of beclin 1, LC3 and p62 in C38 cells on starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (b) Percentage of cells containing more than five GFP–LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus scrambled oligonucleotides; circle, P 0.01 versus CFTR siRNA or CFTRinh-172; ANOVA. (c–g) A549 cells were transfected with 1 μg of pGFP–-CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine. (c) Immunoblot of TG2 and PIASy. (d) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching. (e) Immunoblot of beclin 1 and LC3 in A549 cells after starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (f) Percentage of cells containing more than five GFP–LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001; two asterisks, P 0.01; ANOVA. (g) Immunostaining with anti-p62 (red) and anti-GFP (green) antibodies in starved A549 cells. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. Each experiment was repeated three times. β-Actin was used as loading control for immunoblot. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf5

(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM). (a) Cells were immunostained with anti-HDAC6 (red) and anti-PPAR-γ (green) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (b) FRET analysis of increase in PPAR-γ–Alexa 546 fluorescence after N-CoR Cy5 photobleaching. (c) IB3-1 cells were transfected with either HA-tagged human beclin 1 or the empty vector under nutrient starvation. Cells were immunostained with anti-LC-3 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (d) IB3-1 cells were cultured under nutrient starvation with cystamine (250 μM) in the presence or absence of the PI(3)K inhibitor 3-MA. Immunostaining was with anti-LAMP-1 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (e) IB3-1 cells were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of 250 μM cystamine under nutrient starvation. Cells were immunostained with anti-p62 (red) and anti-CFTR (green) antibodies. Yellow indicates co-localization. N, cell nucleus. Scale bar, 10 μm. (f, g) IB3-1 cells were transfected with 1 μg of pGFP–CFTRF508del or the empty vector, in the presence or absence of 250 μM cystamine. (f) Cells were immunostained with anti-GM-130 (red) and anti-GFP (green) antibodies. Yellow indicates co-localization (arrows). Scale bar, 10 μm. (g) FRET analysis of GFP fluorescence after HDAC6 Cy3 photobleaching. Each experiment was repeated three times. Quantitative measurement of co-localizations is shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf6

(a–d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP–CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides. (a) Left: CFTR revealed with anti-GFP primary antibody in IB3-1 cell lysate. Top arrow, complex-glycosylated form (apparent Mr of band C about 170K, revealed as 210K with GFP tag); bottom arrow, core-glycosylated form (apparent Mr of band B about 150K, revealed as 190K with GFP tag). Uncropped images of blots are shown in Supplementary Information, Fig. S10. Right: quantification of bands B (black) and C (white) normalized to β-actin levels. Values are means ± s.d. for three independent experiments. (b) Cells immunostained with an anti-GFP antibody under non-permeabilizing conditions. Serial confocal sections were collected from top to bottom of cell. (c) Side view (X–Z) of a z-stack of confocal images taken in b. (d) Surface biotinylation assays was performed with membrane-impermeable sulpho-NHS-LC-biotin. The cells were fractioned to obtain the plasma membrane fraction, and biotinylated proteins were precipitated with streptavidin beads. Immunoblot of GFP to reveal the C form. E-cadherin and β-actin were used as positive and negative controls, respectively. (e) IB3-1 cells transfected with either HA-tagged human beclin 1 or empty vector. Immunoblot of PIASy, TG2, phospho-p42/44 and PPAR-γ proteins. (f) IB3-1 cells transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides. Immunoblot of p62 and phospho-p42/44. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (g) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. Immunoblot of phospho-p42/44. (h) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or 50 nM human ATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA. Each experiment was repeated three times. β-Actin was used as loading control. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.