EMBO/soda-nel huggingface dataset:
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Confocal analysis of the subcellular localization of HA–PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm. (b) Immuno-gold EM micrographs of HeLa cells transfected with GFP–PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP–PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.
The phospholipase D1 pathway modulates macroautophagy (2010)
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Confocal analysis of the subcellular localization of HA–PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP–Rab5 (early endosomes), GFP–UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA–PLD1 and GFP–ATG14L (upper panels) or with HA–PLD1 alone (lower panels). Upper panels: HA–PLD1 (red), GFP–ATG14L (green), endogenous LC3 (blue). Lower panels: HA–PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm. (b) Confocal analysis showing lack of co-localization of HA–PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4–C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm. (c) EM analysis showing that GFP–PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4–C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP–PLD1 and pStrawberry-ATG4–C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05. (b) Western blot analysis of HA–PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA–PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA–PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3). (c) Confocal analysis of the co-localization between HA–PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA–PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA–PLD1. (b) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA–PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA–PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA–PXmut). HeLa cells were transiently transfected with a plasmid encoding HA–PXmut and fixed after 2 h of nutrient deprivation. HA–PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180–285 cells for Nm; n=449–519 cells for St). **P0.01. (c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4). (d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4). (c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26–45 for all the conditions). **P0.01; *P0.05.
The phospholipase D1 pathway modulates macroautophagy (2010)
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(a) Immunofluorescent detection of endogenous LC3 (red) in liver sections from fed or starved Pld1 WT and KO mice. Sections were counterstained with DAPI. Scale bar, 5 μm. (b) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice. Histograms show the average number, size and surface area of LC3-positive compartments. Values denote means±s.e.m. ((n=25–28), 4 mice per genotype). **P0.01. (c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.
NAD(P) transhydrogenase has non-mitochondrial vital functions in malaria parasite transmission (2019)
Figure 2

Fig. 2. Expression and subcellular localisation of NTH in P. berghei ookinetes.

A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm.

B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm.

C: Schematic diagram of a genetic cross between parasite lines NTH/GFP and LAP3/mCherry and the resultant ookinete population after mosquito transmission and drug selection (oocysts, liver and blood stage parasites not shown). Colours represent NTH::GFP (green), LAP3::mCherry (red) and their co-localisation (orange). Spots in ookinetes depict crystalloids. Self-fertilization events are omitted.

D: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm.

E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm.

F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.
NAD(P) transhydrogenase has non-mitochondrial vital functions in malaria parasite transmission (2019)
Figure 3

Fig. 3. NTH loss-of-function and contribution to P. berghei parasite development.

A: Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5μm.

B: Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20); ns, not significantly different (Mann-Whitney).

C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.

D: Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20). ** mark statistically significant differences (P<0.0001, Mann-Whitney); ns, not significant.

E: Images of ookinetes of parasite lines NTH∆PP showing dispersed NTH∆PP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; Cross (carrying modified alleles encoding NTH∆PP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5μm.

F: Schematic diagram of a genetic cross between parasite lines NTH∆PP and LAP3/mCherry and the resultant ookinete population after mosquito transmission and drug selection (oocysts, liver and blood stage parasites not shown). Colours represent NTH::GFP (green), LAP3::mCherry (red) and their co-localisation (orange). Spots in ookinetes depict crystalloids. The red cross indicates that sporozoite formation is blocked. Self-fertilization events are omitted.

G: PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH∆PP x LAP3/mCherry genetic cross (lane NTH∆PP), amplifying an approximately 4.4kb fragment. NTH/GFP parasites are used as a PCR control, amplifying approximately 4.6kb. Parental parasites provide a negative control.

H: Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5μm.
NAD(P) transhydrogenase has non-mitochondrial vital functions in malaria parasite transmission (2019)
Figure 4

Fig. 4. Subcellular localisation of NTH in P. berghei oocysts and sporozoites.

A: Brightfield and fluorescence image of an unsporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm.

B: Image of a sporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm.

C: Image of a NTH/GFP sporozoite, showing NTH::GFP localisation in a tubular structure. Scale bar = 10μm.

D: Image of a sporulated oocyst simultaneously expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast. Scale bar = 20μm and 10μm (inset).

E: Image of sporozoites expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast (arrows). Sporozoites expressing only ACP::mCherry or NTH::GFP are also present. Scale bar = 10μm.

F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.

Expanded View Figure Legends
Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart (2019)
Figure 1

Figure 1: Sphinganine accumulation accompanied by loss of genome integrity is an evolutionarily conserved hallmark of the aged vertebrate heart: (A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4). (B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine. (D) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates. (E) Heatmap of differentially expressed transcripts (Log2 fold change +/- 1, adjusted p-value cut off <0.05) between young and aged killifish hearts (n = 3). Significantly enriched relevant GO categories depicted as bar graph. (F) Transcriptional drift variance of genes involved in genome integrity, epigenetic regulation and sphingolipid metabolism. (G) γH2A.X staining of young, mid-aged and aged killifish hearts show signs of DNA damage, increasing with age (n = 4, # of sections per animal = 10 alternate sections, 10 µm thickness). (H) Quantification of γH2A.X+ cardiomyocyte nuclei. (I) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays. Representative images of single nuclei from young and old hearts along with comet length on the right (n = 4). (J) γH2A.X staining of young and aged human ventricles show signs of DNA damage accumulating with age (n = 2, # of sections per sample = 5 alternate sections, 10 µm thickness). (K) Quantification of γH2A.X+ cardiomyocyte nuclei is on the right. Scale bar = 50 µm.
Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart (2019)
Figure 2

Figure 2: Elevated sphinganine level causes genomic and epigenomic aberrations leading to senescence and ageing signatures in human cardiomyocytes: Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition) and (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions. Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right. (K) Proteome analysis confirms sphinganine-induced disruption of (epi)genomic integrity (n = 3). High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.
Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart (2019)
Figure 3

Figure 3: DHS-induced DNA damage in cardiomyocytes is caused by direct inhibition of histone deacetylases: (A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining. (B) Bar-graphs depict quantification of γH2A.X+ hCM nuclei in the indicated conditions. (C) In silico simulation showed docking of sphinganine-analogue DHS1P in the tubular active site of human HDAC1. (D) Sphinganine derivatives DHS1P and S1P show comparable binding affinity to HDAC1, similar to the known HDAC inhibitor TSA. (E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph. (F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs. (G-H) Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications. (I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).
Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart (2019)
Figure 4

Figure 4: SPHK2 and HAT inhibitors prevent the DHS-induced cardiac DNA damage in vitro: (A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs. (C) Bar graphs representing the percentage of γH2A.X+ hCMs in the indicated conditions (# cells quantified per replicate ~200). (D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition). (F) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (G) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels. (H) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (I) HAT inhibition prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) on human cardiomyocytes. (J) Quantification of γH2A.X+ hCMs nuclei represented as percentage bar graph. (K) Curcumin-mediated HAT inhibition lead to inactivation of p21 in hCMs. (L) Violin plots depicting the distributions of the grey-scale nuclear intensity of p21 stained nuclei. (When not specified, the experiments were conducted in at least 3 biological replicates).