EMBO/soda-nel huggingface dataset:
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 6.

Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A–D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A–D): 20 µM; (A′–B′) 2 µM; (C′–D′) 10 µM. (E–G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 7.

Dyn2 associates with autolysosomal tubules. (A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8–27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the lysosomal resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 1

Figure 1: Glia specific loss of ADAM17 results in age-associated retinal degeneration

A. Diagram of a tangential (left) and horizontal (right) section of the adult Drosophila retina, with photoreceptors highlighted in violet and the pigmented glial cells (PGCs) in grey. PR: photoreceptor; R: rhabdomere; CB: cell body.

B-K. Transmission electron microscopy (TEM) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in (B) wild type; (C) ADAM17-/- mutant; (D) ADAM17-/Deficiency; (E, F) RNAi against ADAM17 and control (lacZ), expressed throughout the retina; (G, H) RNAi against ADAM17 and control (lacZ), expressed in the neurons; (I,J) RNAi against ADAM17 and control (lacZ), expressed in the pigmented glial cells; and (K) a kuz-/- mutant.

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from TEM images for the genotypes above, expressed as a stacked bar chart; n=180 ommatidia from 3 different fly retinas for each.

M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red).

N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n=10 fly retinas.

Scale bars: 10μm.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 2

Figure 2: Abnormal LD accumulation in young adult retinas upon a loss of ADAM17 in glial cells

A-C. TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia (A1, B1, C1), or a single ommatidium (A2, B2, C2) in wild type (A1, A2), ADAM17-/- mutant (B1, B2) or ADAM17-/Df (C1, C2). Red arrows show lipid droplets in PGCs, green shading highlights PGCs.

D-J. Fluorescent images of 1 day old fly retinas stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and the photoreceptor membranes respectively; (D) wild type; (E) ADAM17-/- mutant; (F) ADAM17-/ Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz-/-; n=10 fly retinas.

K-L. Quantitation of the BODIPY signal shown as integrated total lipid and normalised LD size of lipid droplets for the genotypes in (D-I). The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.

M-N. Q-PCR analysis of mRNA transcripts of lipogenic genes -Acetyl CoA carboxylase (ACC) and Fatty Acid Synthase (FASN) from heads of wild type and ADAM17-/- mutants; n=4 and n=3 biological replicates for M and N respectively, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.

Scale bars for A2, B2 and C2: 2μm and 10μm for all other panels.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 3

Figure 3: Overexpressing lipase in ADAM17 mutant retinas rescues both lipid droplet accumulation and retinal degeneration

A-D. Fluorescent images of 1 day old retinas, stained with BODIPY (green) and FM-dye (red) to stain lipid droplets and the photoreceptor membranes respectively; (A) wild type; (B) ADAM17-/- mutant; (C) overexpression of lipase in glial cells of ADAM17-/- mutant; (D) overexpression of lipase in neurons of ADAM17-/- mutant.

E-G. Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size; (G) integrated total lipid, for the genotypes mentioned above, n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier.

H-K. TEM images of 5 week old adult retinas corresponding to (H) wildtype; (I) ADAM17-/- mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17-/-mutant.

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above, presented as a stacked graph plot; n=180 ommatidia from 3 different fly retinas for each.

Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.

Scale bars: 10μm.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 4

Figure 4: Loss of either Eiger or Grindelwald, but not Wengen, leads to abnormal LD accumulation and age-associated retinal degeneration.

A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively.

D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-.

I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier.

J-K. mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type, eiger-/- and grindelwald-/- . n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean

L-O. TEM images of two week old retinas corresponding to (L) control; (M) eiger-/-; (N) grindelwald-/-; (O) wengen-/-. n=180 ommatidia from 3 different fly retinas for each. P. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from 2 week old retinas observed in TEM, corresponding to the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each.

Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.

Scale bars: 10μm.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 5

Figure 5: Reducing mitochondrial ROS levels in ADAM17-/- mutants leads to a rescue of the LD accumulation and retinal degeneration.

A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4.

E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.

F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4.

J. Percentage of normal, abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each.

K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant.

O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01, *p<0.05.

P-Q. TEM analysis of 5 week aged retinas corresponding to either ADAM17-/- mutants or SOD2 over-expression in an ADAM17-/- mutant.

R. Percentage of normal, abnormal and missing rhabdomeres in ADAM17-/- mutant retinas, as compared to retinas overexpressing glial specific SOD2 in an ADAM17-/- mutant background observed with TEM; n=180 ommatidia from 3 different fly retinas for each.

S. Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17-/-.

T. Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.

Scale bars: 10μm.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 6

Figure 6: Activity dependence and role of JNK in driving LD accumulation and retinal degeneration in ADAM17-/- mutants.

A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark.

C-F. TEM images of 4 week old retinas: (C, D) wildtype, or (E, F) ADAM17-/- flies reared from first larval instar either in total light (C, E) or total dark (D, F).

G. Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A-B, all reared in the dark. n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

H. Percentage of normal, abnormal and missing rhabdomeres in the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each.

I. Measurement of MDA levels (in nmoles) from 1 day old retinas of wild type or ADAM17-/- , reared either in light or dark. Four biological replicates with 3 technical replicates for each genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean.

K-L. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) heterozygous mutant of bsk alone; or (L) in combination with ADAM17-/-.

M. Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K-L, along with w1118 and ADAM17-/-. n=10 flies per genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

N-O. TEM images of 2 week old adult retinas, showing clusters of ommatidia for a heterozygous mutant of bsk alone (M) or in combination with ADAM17-/- (N); n=3 for each.

P. Percentage of normal, abnormal and missing rhabdomeres for the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each.

Data were quantified for significance using Student's t test. **p<0.01.

Scale bars: 10μm.
ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration (2020)
Figure 7

Figure 7: Inhibiting ADAM17 activity in human iPSC derived microglia cells leads to abnormal LD phenotypes.

A-C. Fluorescent images of human iPSC derived microglia cells, labelled with 493/503 BODIPY (green) and FM-dye (red) to mark lipids and membranes respectively. iPSC derived microglia cells were obtained from 3 different donors, and treated with either DMSO, GW, TAPI-1 or GI.

D-E. Quantitation of the BODIPY signal shown as integrated total lipid and normalised LD size (normalised to DMSO) for cells corresponding to each treatment and donor mentioned in A-C. n=15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.

F-G. Q-PCR analysis of mRNA transcripts of lipogenic genes FASN and LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO, GW or GI for 24 hours. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean.

H-J. Fluorescent images of Mitotracker and MitoSOX labelling of cells from Donor number 2 treated for 24hours with either DMSO, GW or GI.

K. Quantitative measurements of MitoSOX intensity normalised to that of Mitotracker in iPSC derived microglia cells from three different donors, treated with either DMSO, GW or GI; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

L-N. Fluorescent images of C-11 BODIPY treated microglia cells from Donor no.1, treated for 24 hours with either DMSO, GW or GI. These representative images display relative amounts of peroxidated and non-peroxidated lipids.

O. Quantification of BODIPY C-11 staining, shown as ratios of peroxidated versus non-peroxidated lipids, depicted as normalised intensity; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean.

Apart from the data in panels D and E, all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.

Scale bars: 10μm
Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. (2009)
Figure 1.

LC3 and p62 accumulate in IBMPFD muscle tissue. (A and B) Immunoblot of 9-mo-old quadriceps muscle lysates from control (cont) or VCP-WT, -RH9, or -RH12 mutant transgenic lines with p62 (A) or LC3 (B) antibodies. Note the increase in p62 and LC3II isoforms in mutant animals. Densitometric quantification is from more than six 9-mo-old animals/group. LC3 and p62 levels are normalized to loading control. Error bars represent the standard error from six independent experiments. **, P < 0.001. AU, arbitrary unit. (C) p62 immunostaining of tibialis anterior (TA) or quadriceps (Quad) muscle from 9-mo-old control, VCP-WT, or one of two VCP-RH transgenic lines (RH9 or RH12). Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions. (D) Histochemistry and immunohistochemistry of quadriceps muscle from VCP-RH–expressing transgenic mice. Hematoxylin and eosin (H&E) staining of a 15-mo-old animal with an RV, p62 immunostaining of an RV from a 12-mo-old animal, LC3 immunostaining of an RV, and subsarcolemmal accumulations of LC3 from a 12-mo-old animal. The bracket highlights one LC3-positive myofiber. A single muscle fiber is outlined in white. (C and D) Arrows denote vacuoles or accumulations of p62 or LC3. Bars, 30 µm.
Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. (2009)
Figure 2.

LC3 and p62 accumulate in VCP-KD and IBMPFD mutant–expressing cells. (A) Lysates from scrambled siRNA or VCP-targeted siRNA–treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. (B) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted. (C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells. (D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A–C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.
Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. (2009)
Figure 3.

Functional VCP is required for autophagosome maturation. (A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD– or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001. (D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)–expressing cells. Bars, 15 µm.