EMBO/soda-nel huggingface dataset:
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 4

Figure 4. α-synuclein fibrils induce TNTs formation and use TNTs to efficiently spread between neuron-like CAD cells.

(A) Representative images of control cells (upper panel) or cells treated with α-synuclein fibrils ATTO-550 (bottom panel) for 16 hours; in red: α-synuclein fibrils and in green: WGA Alexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).

(B) Relative percentage of TNT-connected cells after α-synuclein fibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student’s t-test).

(C) Confocal images showing one TNT connecting two cells loaded with α-synuclein fibrils ATTO-550 (in red) 16 hours after co-culture and stained with WGA Alexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synuclein fibrils (red) are present in the lumen of the TNT (WGA Alexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets).

(D) Confocal image showing TNT connecting a donor cell containing α-synuclein fibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synuclein fibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synuclein fibrils in the lumen of the TNT; in red: α-synuclein fibrils, in green: GFP transfected acceptor cells in blue: WGA Alexa-488. Scale bars represent 10 μm and 3 μm for the insets.

(E) Relative percentage of TNTs in cells containing α-synuclein fibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs.

(F) Quantification of the average number of ATTO-550 α-synuclein fibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synuclein fibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiI vesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).

(G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student’s t-test).

(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synuclein fibrils transfer compared to condition allowing TNT formation (sub-confluent condition).
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 6

Figure 6. α-synuclein fibrils are found in lysosomes and are efficiently transferred between primary neurons

(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm.

(B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.

(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.

(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.

(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.

(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 7

Figure 7. Cell-to-cell contact facilitates α-synuclein transfer between primary neurons

(A) Experimental set-up used to assay transfer through conditioned medium (upper scheme) and cell-to-cell contacts (lower scheme) in neurons. For the CM experiments, donor neurons are loaded with α-synuclein fibrils for 16 hours, washed 3 times with PBS and then fresh medium is added and left to condition for 72 hours. CM is then collected and transferred to acceptor neurons (10 div) for 72 hours. In the cell-to-cell contact impairment (No contact) experiment the donor (already loaded) and newly dissected acceptor (already labelled) neurons are plated on different coverslips placed in the same Petri dish. In both experiments, the percentage of cells containing α-synuclein ATTO-550 puncta, and the number and mean size of α-synuclein puncta per cell are quantified using the ICY software after 72 hours.

(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synuclein fibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.

(C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student’s t-test. Data show mean ± s.e.m from 3 independent experiments.

(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments)..

(E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 8

Figure 8. Transfer of α-synuclein fibrils between neurons and neuron-like CAD cells.

(A) Representative images of donor (upper panel) and acceptor cells (bottom panel) after 24 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with H2B-GFP-acceptor CAD cells. In red: α-synuclein puncta, in green: acceptor CADs, in white donor neurons and in blue: nuclei. The images of donor cells are representative Z-stack projections of the lower slices where these cells are located, and the images corresponding to acceptor CADs are Z-stack projections of the upper slices, covering the whole cell body. Scale bar represents 10 μm.

(B) Box plots showing the number (left side) and average size (right side) of α-synuclein puncta in acceptor CAD cells after 24 hours in co-culture with donor neurons. (n=3 independent experiments).

(C) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co-culture. In green: MAP-2, in white: WGA, in red: α-synuclein puncta and in blue nuclei. The merge image in the bottom panel shows in detail the TNT connecting the cells, and the insets and the 3D reconstruction of the neuron (green) and the CAD cell (white) show the presence of α-synuclein puncta inside the TNT.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf1

(a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice. Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum (Fed). Bars represent mean ± s.d. for
n = 5 mice;
*P≤0.05;
**P≤0.01;
***P≤0.001. (b) Representative β-gal staining of liver and kidney frozen sections isolated from fed and 24-h-fasted heterozygous Tcfeb– β-gal mice. (c) Time-course expression analysis of TFEB in wild-type HeLa cells, MEFs and hepatocytes after the addition of starvation media (time 0). Data represent mean ± s.d. for
n = 3 independent experiments. (d) Expression levels of transfected hTFEB–FLAG, Tcfeb– β-gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control (+/+) and heterozygous Tcfeb– β-gal/+ mice. Data represent mean ± s.d. for
n = 3 mice;
*P≤0.05. (e) Time-course expression analysis of TFEB mRNA in control (Ctrl) or
TFEB-overexpressing HeLa cells during fasting and re-feeding. Primers specific for the endogenous TFEB were used. Data represent mean ± s.d. for
n = 3. (f) Time-course expression analysis of Tcfeb mRNA in heterozygous Tcfeb– β-gal/+ or control MEFs during fasting and re-feeding. Specific primers for the endogenous Tcfeb were used. Data represent mean ± s.d. for
n = 3 mice. (g) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb– β-gal/+ or control hepatocytes in fed and after 24 h fasting. Data represent mean ± s.d. for
n = 3 mice. (h) ChIP analysis from liver of mice fed ad libitum or 24 h fasted. The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1. Red rectangles represent exons, and the ATG indicates the first codon (from the mouse Tcfeb isoform b). The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Data represent mean ± s.d of 3 independent experiments.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf3

(a) ChIP analysis from liver of mice fed ad libitum (fed) or 24 h-fasted (starved). CLEAR sites in the promoter region of Pgc-1α are indicated by squares. Numbers indicate the distance (bp) of the binding element from the start codon. Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Bar graphs represent mean ± s.d of 3 independent experiments,
*P≤0.05;
**P≤0.01. (b) Representative diagrams of the constructs containing the promoter region of Pgc-1α with either intact (Pgc-1α WT) or deleted (Pgc-1α DEL) CLEAR elements upstream of the luciferase cDNA. (c) Luciferase activity was measured after transfecting increasing amounts of TFEB–FLAG in combination with Pgc-1α WT or Pgc-1α DEL plasmids. Bar graphs represent mean ± s.d. of
n = 3independent experiments,
****P≤0.0001 compared with mock transfected cells. (d) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media. Bar graphs represent mean ± s.d. of
n = 3independent experiments
**P≤0.01 compared with mock transfected cells. (e) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control, HDAd-TFEB and
Tcfeb-LiKO (liver KO) mice treated as indicated. Bar graphs show mean ± s.d. for
n = 4 mice.
*P≤0.05;
**P≤0.01. (f) Expression analysis of TFEB target genes in liver of mice with indicated genotypes. Bar graphs show mean ± s.d. for
n = 3 mice.
*P≤0.05;**P≤0.01;
***P≤0.001. (g) Expression analysis of PGC1α/PPARα target genes in liver from either fasted or fed mice with indicated genotypes. Bar graphs show mean ± s.d. for
n = 4.
*P≤0.05;
**P≤0.01 compared with the respective controls (fed or fasted).
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf4

(a) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted. Original magnification ×40. (b) Toluidin blue staining of liver sections isolated from fed and 24-h-fasted Tcfeb-LiKO and control mice (Tcfeb flox/flox mice). Arrows indicate lipid droplets. Original magnification ×100. (c) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis. A representative image is shown on the right. Values are mean ± s.d. of at least 10 cells per mouse (n = 3 mice per group).
*P≤0.05. (d) The oxygen consumption rate (OCR) in primary hepatocytes isolated from control and Tcfeb-LiKO mice was measured with an XF24 analyser (Seahorse) before and after the addition of palmitic acid (0.2 mM) conjugated with BSA. The vertical red line indicates the time at which palmitate was added to cells. Values are mean ± s.d. for 3 independent experiments
*P≤0.05. (e,f) Total FFA (e) and glycerol (f) in the serum isolated from 6-h-fasted Tcfeb-LiKO and control mice. Values are mean ± s.d. (n = 5 mice per group)
*P≤0.05 compared with controls. (g) Total serum ketones in fed and fasted Tcfeb-LiKO and control mice. Bars are mean ± s.d. for
n = 10 mice per group.
**P≤0.01 compared with fed control mice. (h) EchoMRI measurement of fat and lean mass in fed and in 24- and 48-h-fasted mice expressed as relative percentage to fed (100% in the graph). Indicated values are mean ± s.d. for
n = 5 mice per group.
*P≤0.05;**P≤0.01 compared with fed control mice. (i) Visceral fat pad mass isolated from 2-month-old-mice with indicated genotypes. Indicated values are mean ± s.d. for
n = 5 mice per group.
*P≤0.05; compared with fed control mice.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf5

(a) Mice with the indicated genotype were kept on a high-fat diet (HFD) for 12 weeks when indicated. Gross liver morphology (upper panel), haematoxylin–eosin (H/E) (middle panel), and oil red O staining of liver sections (bottom panel). Ctrl, control. (b,c) Bar graph showing normalized liver weights (mean ± s.d. for
n = 10 mice per group; b) and total lipid content in mice with indicated genotype (mean ± s.d. for
n = 10; c).
*P≤0.05;***P≤0.001 compared with control. (d) Toluidin blue staining of liver sections isolated from Atg7-LiKO mice injected with HDAd-Ctrl or HDAd-TFEB vector. (e,f) Bar graph showing normalized liver weights (mean ± s.d. for
n = 10 mice per group
***P≤0.001; e) and total lipid content in mice with the indicated genotype (mean ± s.d. for
n = 5 mice per group;
***P≤0.001; NS, not significant; f). Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf6

(a,b) Body weight (a) and visceral fat mass (b) isolated from 2-month-old-mice with the indicated genotypes.
n = 5 mice per group. (c–i) Serum metabolic profile in HDad-TFEB mice compared with control mice. (j,k) Respiratory exchange ratio (RER; VCO2/VO2; j) and fatty acid utilization calculated from data in j (k). Values are mean ± s.d (n = 10 mice per group)
*P≤0.05;**P≤0.01; compared with controls. VLDL, very-low-density lipoprotein; HDL, high-density lipoprotein.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf7

(a) Body weight curves of male mice fed with the high-fat diet (HFD; 40% calories from fat) for 10 weeks starting from 5 weeks of age (0 on the
x axis). Mice were injected with HDAd-TFEB either 1 week before (early inj), or 4 weeks after, (late inj) being placed on the high-fat diet, as indicated by the arrows. Values are represented as percentages of weight increase. Ctrl, control. (b) Whole-body composition analysis (EchoMRI) of the same mice as in a after 10 weeks of the high-fat diet. In a,b
n = 10 mice per group; bars represent mean ± s.d.
*P≤0.05;**P≤0.01;
***P≤0.001 compared with control high-fat diet group. (c–f) Total serum insulin, leptin, triglyceride and cholesterol levels in control and HDAd-TFEB mice kept on the high-fat diet for 10 weeks. Value are mean ± s.d.
n = 10 add per group.
*P≤0.05;
****P≤0.0001 compared with control. (g–i) Glucose and insulin tolerance tests in control and HDAd-TFEB mice challenged with the high-fat diet for 10 weeks. (g,h) Glucose (g) and serum insulin (h) levels at the indicated time points after glucose challenge. (i) Glucose levels at the indicated time points after insulin challenge. In g–i, values are mean ± s.d.
n = 7 mice per group;
*P≤0.05;**P≤0.01 compared with control. Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop (2013)
figf8

(a) hlh-30 quantitative rtPCR showing increased expression of the C. elegans TFEB gene hlh-30 over a time course of starvation in wild-type animals followed by a rapid decrease to the basal level following re-feeding of the animals (mean±s.e.m. of
n = 3).
*P≤0.05 (t-test, compared with wild-type at
t = 0 h). (b) hlh-30 3’ UTR quantitative rtPCR showing expression of hlh-30 after 12 h starvation in wild-type and hlh-30(tm1978) animals (mean±s.e.m. of
n = 3).
*P≤0.05 (t-test, compared with wild-type starved). (c) Quantification of oil red O stain in starved animals relative to well-fed counterparts (mean±s.e.m. of
n = 3).
**P≤0.01 (t-test, compared with wild-type starved). (d–g) Representative micrographs of wild-type and hlh-30 animals after 8 h starvation and stained with oil red O. (h–k) Representative TEMs of wild-type and hlh-30 animals after 24 h starvation. IEC, intestinal epithelial cell; EPI, epidermis; BB, brush border; GON, gonad; BWM, body wall muscles, aj, apical junction; ld, lipid droplet. Scale bar, 2 μm. (l) Quantitative rtPCR of starvation-induced genes in wild-type and hlh-30 animals after 12 h starvation (means ± s.e.m of
n = 3).
*P≤0.05;
**P≤0.01;
***P≤0.001 (t-test, compared with wild-type starved). (m,n) hlh-30 is required for starvation-induced lifespan extension. One representative experiment of 2 independent trials; error bars represent mean±s.e.m. Median survival (MS) (wild-type fed)   =  10 d; MS (wild-type starved)   =  17 d,
P0.0001 versus fed using the Log-rank test; MS (hlh-30 fed)   =  7 d; MS (hlh-30 starved)   =  9 d,
P0.0001 versus fed. (o) L1 arrest assay showing survival of wild-type and hlh-30 starved animals relative to non-starved conditions (mean±s.e.m. of
n = 3).
***P≤0.001 (t-test, compared with wild-type starved).
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 1

Figure 1. BRCA2 physically interacts with DDX5

(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control (cropped image is shown).

(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.

(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.

(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control (cropped image is shown).

(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 2

Figure 2. DDX5 depletion leads to a genome-wide accumulation of DNA-RNA hybrids particularly enriched at DSBs

(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments. See also Fig EV2B.

(B) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. See also Figure EV2C.

(C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments. See also Fig EV3.

(D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated.

(E) DNA-RNA hybrid metaplot distribution over γH2AX ChIP-seq peaks. Peak metaplot shows the mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC).

(F) Venn diagram representing the overlap between γH2AX-positive genes in K562 cells (γH2AX ChIP-seq) and genes that specifically accumulate hybrids in control cells (top) or in DDX5-depleted cells (bottom).