EMBO/soda-nel huggingface dataset:
Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection (2021)
Figure 5

Figure 5. MOV10 and SPN are included in the viral particle, whereas SERINC3 is excluded from the virus and present in virus-modified EVs

(A, B) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD).

(C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom).

(D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets).

(F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments.

(G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown.

(I) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.
Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection (2021)
Figure 6

Figure 6. SERINC3 down-regulation modifies the EVs released by Jurkat cells.

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes).

(A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown.

(B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells. *P<0.05, **P<0.01 (One way ANOVA followed by Dunnett's post-test). Mean ± SD of three independent experiments is shown.

(C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom).

(D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets).

(E, F) 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E) and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells.

(G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM. Mean ± SD of three independent experiments is shown. *P<0.05, **P<0.01, ***P<0.001 (One way ANOVA followed by Dunnett's post-test).

TABLES

Table 1: Classified list of proteins with similar profiles to HIV-1 Gag p24 using parameter cut offs Min ratio count: 1, Min correlation of replicates: 0, Min F3 enrichment: -1.

Table 2: Proteins with strong modification of EV-associated secretion (M score > 1) with high reproducibility (R > 0.9) upon HIV infection of Jurkat cells (top-right quadrant of Fig 4C). Estimated FDR = 8%.
Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns (2020)
Figure 1

Figure 1: Establishment of FANS for stem cells of the shoot apical meristem (SAM).

(a) Expression of H2B-mCherry under control of the CLV3 promoter in 14-d-old seedlings. Whole-mount immunostaining using α-mCherry antibodies and laser scanning microscopy (scale bar 10 μm).

(b) Example of a FANS experiment: mCherry-positive (+) and mCherry-negative (-) gates of DAPI-gated nuclei. Numbers indicate total number and percent of DAPI (for -) and mCherry (for +) events.

(c) A representative example for enrichment of CLV3 transcript in mCherry-positive nuclei determined by qRT-PCR and normalized to wt (N = 1).
Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns (2020)
Figure 2

Figure 2: Differential RNA expression in SAM stem cells during development.

(a) Expression of CLV3, mCherry, TEL2, PAN, and the meristem marker genes STM and KNAT1. Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2). + = stem cell nuclei; - = non-stem cell nuclei, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.

(b) Global gene expression correlations at different developmental stages of stem (+) and non-stem cell (-) nuclei.

(c) Number of DEGs between stem and non-stem cells at each timepoint. The banded portion of the bars indicates the number of transcription factor genes (also in parenthesis).

(d) Overlap of genes with higher expression in stem cells (excluding mCherry) and significant GO-terms for shared DEGs (increased expression during at least 3 timepoints). See also Appendix Fig. S2 for p-values of overlapping gene sets. p-values were calculated with Fisher's exact test and Bonferroni-corrected (see also Appendix Table EV3).
Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns (2020)
Figure 5

Figure 5: Differential expression of individual TEs.

(a) Increased expression of TEs in stem cells of D7 seedlings according to qRT-PCR. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR. N = 2 (ATLANTYS3, ATCOPIA39, ATCOPIA49, ATCOPIA29, ATGP1-1), N = 3 (VANDAL6, ATGP2, ATGP1-2, ATLINE1, ATGP1-3), N = 4 (ATHILA6A, ATCOPIA83), N = 5 (VANDAL12, ATHILA3, ATCOPIA22), and N = 6 for CLAVATA. *p<0.01, **p<0.001 (t-test).

(b) Heatmap of TE expression in several silencing mutants determined by qRT-PCR relative to a housekeeping gene. To be able to display data in log-scale also for samples with zero expression, a value of 1.0e-6 (factor 10 smaller than the smallest value) was added to all values. Data for nuclei and CLV3::H2BmCherry are the same as in (a). For seedling samples, N = 2 (ATCOPIA49, ATHILA3), N = 3 (ATCOPIA39, VANDAL6, ATGP2, ATLINE1, ATCOPIA29, ATGP1-1), and N = 4 (ATLANTYS3, VANDAL12, ATGP1-2, ATHILA6A).
Calcium signals regulate the functional differentiation of thymic iNKT cells (2021)
Figure 2

Figure 2. NKT2 cells require more Ca2+ for proliferation and differentiation

(a) Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown.

(b) Cell proliferation was quantified as division index based on CTV signals in (a).

(c-i) Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. (c,d) EGTA was added from the start of culture (day 0), and the percentages of CD4+ T cells (c) and iNKT cells (d) among live cells are shown. (e-i) EGTA was added 48h after the start of culture (day 2), and the percentage of iNKT cells (e), the percentage of Tbethi population in iNKT cells (f), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.

Data information: Data are representative from one of at least three independent experiments. Graphs represent mean ± SD with three replicate samples (b) or symbols representing individual thymic lobes (c-i). For (b), ****p < 0.0001; n.s. not significant (nonparametric Spearman correlation between division index and EGTA concentration); For (c-i), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).
Calcium signals regulate the functional differentiation of thymic iNKT cells (2021)
Figure 4

Figure 4. Mitochondria in NKT2 support robust oxidative phosphorylation.

(a) MitoTracker green staining in iNKT cell subsets ex vivo;

(b) Quantification of mean fluorescence intensity (MFI) of MitoTracker green staining in iNKT cell subsets;

(c) Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.

(d) Mitochondrial volume estimated by Tim 23 staining as in (c), and presented as % of total cell volume;

(e) Representative images of electron microscopy in NKT1 and NKT2 cells;

(F, G) (f) individual mitochondria size and (g) cristae perimeters normalized to mitochondria perimeter based on EM in (e).

Data information: Data are representative from three independent experiments. Graphs represent mean ± SD with symbols representing individual mice (b), individual cells (d), or individual mitochondrion (f, g). *p < 0.05; ***p < 0.001; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).
Calcium signals regulate the functional differentiation of thymic iNKT cells (2021)
Figure 5

Figure 5. ORAI is required for iNKT cell subset differentiation.

(a) Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);

(b) Percentage of iNKT cells in total live cells in FTOC with or without BTP2 (1uM) addition at the start of culture (day 0);

(c-f) BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet (c), CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf1

(a–d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a) or treatment with rapamycin (rapam) or thapsigargin (thapsig) (b). (c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl. (d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA). (e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli. (f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference. (h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf2

(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (−) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4. (b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4. (c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4. (d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c. (e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf3

(a) Electron micrographs of striatal neurons from control and HD94 mice grown over a wild-type rat astrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13–17 cell profiles in triplicate experiments; *P 0.05). Arrows, enlarged electron-clear vesicles. (b) Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice. (c) Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles. (d) Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf4

(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles). (b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf5

(a) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4. (b) Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P 0.05. Full-length blots in Supplementary Figure 20.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf6

(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values. (b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter. (c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel. (d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a. (e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf7

(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503. (b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3. (c–e) Neutral lipids in striatal cells from 7Q-htt and 111Q-htt mice (c), primary striatal neurons from 18Q-htt and 111Q-htt mice grown on a monolayer of their own astrocytes (d) and lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b. (f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs. (g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05. (h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel. (i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.
Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease (2010)
figf8

(a) Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10–20 cells in three different experiments. (b) Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type rat astrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates. (c) Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in b. Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean. (d,e) Striatal cells from 7Q-htt and 111Q-htt mice (d) and MEFs from 18Q-htt and 111Q-htt mice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05. (f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f) or BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.
Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. (2013)
Figure 1.

Epg5−/− mice show motor deficits. (A) Scheme for generating Epg5 knockout mice. HSVTK, herpes simplex virus thymidine kinase; Neo, neomycin. (B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot. (C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown. (D) Time spent on a rotarod. The horizontal lines indicate mean times. (E) Epg5−/− males show gradually worsening limb-clasping reflexes. (F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age. (G) Spinal deformation in an Epg5−/− male at 10 mo of age. (H) Epg5−/− females gradually develop the limb-clasping phenotype.