EMBO/soda-nel huggingface dataset:
The ubiquitin ligase parkin mediates resistance to intracellular pathogens (2013)
f4

a, WT and Park2−/− mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P  0.02 by Student’s t-test). b, Enumeration of liver and spleen c.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P  0.03 by Student’s t-test). c, Survival of M. tuberculosis-infected wild-type and Park2−/− mice (n = 10, **P  0.001 by log-rank test). d, Immunohistochemistry staining of lung sections from infected mice 21 days post-infection, scale bar, 50 &mgr;m. e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 &mgr;m (×4), 50 &mgr;m (×40). f, Confocal microscopy images of human lung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 &mgr;m. g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P  0.04 by Student’s t-test).
The ubiquitin ligase parkin mediates resistance to intracellular pathogens (2013)
f5

a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting. b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3–5, **P  0.001 by Student’s t-test). c, Survival of D. melanogaster was determined (n = 33, **P  0.001 by log-rank test). d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3–5 per group, **P  0.009 by Student’s t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test). f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 1

Figure 1. Hyperactivity of Parp1 in Xrcc1Nes-Cre brain.

[a] Sagittal sections obtained from mice (p15) of the indicated genotypes were immunostained for ADP-ribose using the pan-ADP-ribose detection reagent MABE1016. Representative images showing levels of ADP-ribose in the hippocampal regions CA1, CA3, and dentate gyrus (DG), and in the cerebral cortex. Red dotted boxes highlight the elevated ADP-ribose staining in Xrcc1Nes-Cre cerebellum (left dotted box) and hippocampus (right doted box). Scale bars: 5 mm, 50 μm. WT (n = 4 mice), Xrcc1Nes-Cre (n = 4), Parp1+/- /Xrcc1Nes-Cre (n = 4), Parp1-/-/Xrcc1Nes-Cre (n = 3) and Parp1-/- (n = 3). Summary histograms show mean ± SEM. Pairwise comparisons between WT versus Xrcc1Nes-Cre mice and WT versus Parp1-/-/Xrcc1Nes-Cre mice were conducted by Kruskal-Wallis ANOVA with Dunn's post-hoc test and statistically significant differences (*p<0.05) are shown.

[b] Protein extracts from wild type (WT), Xrcc1Nes-Cre, and Parp1-/- forebrain tissue, containing hippocampus and cortex, were incubated with 1 mM NAD+ for 45 min in the presence or absence of PARP inhibitor as indicated, and ADP-ribosylation detected by western blotting using the poly(ADP-ribose)-specific detection reagent MABE1031. Representative images from two or more independent experiments are shown. A western blot showing the level of Parp1 and Xrcc1 in the forebrain tissue extracts is also shown, Inset.

[c] Protein extracts from wild type (WT) and Xrcc1Nes-Cre forebrain or cerebellum were incubated for 0, 5 ,10 ,15, 30 & 45 min in the presence of NAD+ as above.

[d] NAD+ levels in wild type (WT), Xrcc1Nes-Cre, and Parp+/-/Xrcc1Nes-Cre forebrain tissue. Data are the scatterplots of individual measurements from at least three mice per genotype, with error bars representing the mean +/-SEM. Statistically significant differences are indicated (Kruskal-Wallis ANOVA with Dunn's post hoc test *p < 0.05).
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 2

Figure 2. Parp1 hyperactivation triggers juvenile seizures and mortality in the absence of Xrcc1.

[a] Kaplan-Meier curves for survival of mice of the indicated genotypes. The table shows number of individuals in each group, median survival (weeks), and p-values from pairwise curve comparisons; Und: undetermined (Log-rank Mantel-Cox tests).

[b] Video monitoring and recording of generalised running/bouncing seizures in mice of the indicated genotypes from P15-P19. The point of death of Xrcc1Nes-Cre mice by fatal seizure is indicated (cross). Note no seizures from mice of other genotypes were detected. WT (n = 9 mice), Xrcc1Nes-cre (n = 3 mice), Parp1+/- /Xrcc1Nes-Cre (n = 3 mice), and Parp1-/-/Xrcc1Nes-Cre (n = 3).
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 3

Figure 3. Suppression of seizure-like activity in Xrcc1Nes-Cre brain slices by Parp1 deletion.

[a] Plots of enveloped spike activity waveforms based on targeted extracellular recordings from hippocampal CA3 region perfused into epileptogenic buffer for the indicated genotypes. Upward deflections indicate seizure-like episodes (SLEs). Scale bars, 5% of maximum amplitude.

[b] Heatplots of cumulative SLEs: WT (n=11 slices from 3 mice), Xrcc1Nes-Cre (n=12 slices from 4 mice), Parp1+/-/Xrcc1Nes-Cre (n=12 slices from 3 mice) and Parp1-/-/Xrcc1Nes-Cre (n=10 slices from 3 mice). Each horizontal bar corresponds to one slice with the colour code indicating cumulative SLEs.

[c] Summary of mean ± SEM cumulative SLE counts. The number of replicates was the same as in (b).

At the recording endpoint, the total SLE count in Xrcc1Nes-Cre is significantly higher than both WT and Parp1-/- Xrcc1Nes-Cre (Kruskal-Wallis ANOVA, p < 0.0017, * indicates significance with Dunn's post-hoc comparisons).
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 4

Figure 4. Suppression of seizure-like activity in Xrcc1Nes-Cre brain slices by PARP1 inhibitor.

[a] Brightfield image (left) of an acute brain slice positioned on HD-MEA with targeted regions indicated. (Right) progression of a typical seizure event in a slice perfused with epileptogenic buffer; colour-code indicates voltage changes in microvolts.

[b] Representative traces from four channels in the CA3 region of hippocampus recorded from 10-15 min in epileptogenic buffer, showing seizure-like activity in Xrcc1Nes-Cre. Vertical scale bar indicates 300 μV.

[c] Mean cumulative activity plots in CA1 and CA3 regions of hippocampus and cortex over 15 min of recording in epileptogenic buffer. WT (n=8 slices from 4 mice), Xrcc1Nes-Cre (n=9 slices from 4 mice).

[d] Summary histograms of the mean (± SEM) cumulative seizure-like activity at 15 min, from the data in panel c. Statistical significance was assessed by ANOVA with post hoc pairwise comparisons (*p < 0.05).

[e] Summary histograms (mean ± SEM) of cumulative seizure-like activity at 5, 10, and 15 min timepoints in cortex, CA1, and CA3 regions of wild type and Xrcc1Nes-Cre brain slices from mice treated or not for 5-8 days ad libitum with PARP1 inhibitor (ABT-888) prior to analysis. WT (n = 8 slices from 4 mice), Xrcc1Nes-Cre (n = 10 from 5 mice), WT + PARPi (n = 6 from 3 mice), Xrcc1Nes-Cre + PARPi (n = 9 from 3 mice). Pairwise comparisons at the 15 min time point between WT versus Xrcc1Nes-Cre mice and WT versus Xrcc1Nes-Cre mice + PARPi were conducted by Kruskal-Wallis with Dunn's post-hoc tests and statistically significant differences (*p<0.05) are shown.
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 5

Figure 5. Parp1 hyperactivation in isolated Xrcc1Nes-Cre hippocampal neurons.

[a] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose (red), NeuN to identify neurons (green), and counterstained with DAPI (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm.

[b] Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90), and Parp1-/-/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal-Wallis ANOVA, p = 0.0013 and Dunn's post-hoc tests).
Parp1 Hyperactivity Couples DNA Breaks to Aberrant Neuronal Calcium Signalling and Lethal Seizures (2021)
Figure 6

Figure 6. Aberrant presynaptic calcium signaling in Xrcc1-/- hippocampal neurons is rescued by Parp1 deletion or inhibition.

[a] Representative image of DIV15 dissociated cultured neurons used. Scale bar: 100 µm.

[b] (top) Cartoon schematic illustrates SyGCaMP6f targeting and action. (bottom) Image shows typical punctate SyGCaMP6f expression at DIV15. Scale bar, 20 µm.

[c] Representative images of fluorescence responses in synaptic terminals expressing SyGCaMP6f to a train of 10 stimuli at 10 Hz in dissociated hippocampal neurons derived from wild type and mutant mice. Scale bar: 5 µm.

[d,e] Mean SyGCaMP6f responses to three rounds of 10 APs stimulation from mice of the following genotypes; WT (n = 1946 synapses, 9 coverslips, 3 animals), Xrcc1Nes-Cre (n = 3313, 12, 4), Parp1+/- /Xrcc1Nes-Cre (n = 2272, 9, 3), and Parp1-/- /Xrcc1Nes-Cre (n = 2122, 11, 3).

[f] Response to chronic treatment with the PARP inhibitor, KU 0058948 Hydrochloride (1 µM) for 9-11 days prior to imaging for Xrcc1+/+ (n = 1264 synapses, 8 coverslips, 3 animals) and Xrcc1Nes-Cre (n = 2231, 10, 4) mice.

[g] Summary histogram of the mean (± SEM) of the integrated fluorescence responses for all individual synapses for each condition. Synapse number is the same as in (D-F). Responses are significantly higher in Xrcc1Nes-Cre synapses versus both WT and Parp1-/-/Xrcc1Nes-Cre (one-way ANOVA, *p < 0.0033, and pairwise student's t-tests).

Data information: (D-F) Data are from three or more independent experiments per genotype/treatment type, combined into one experimental data set and plotted on three separate graphs for clarity. Dashed lines in panels e and f are the WT and/or Xrcc1Nes-Cre curves transposed from panel d for comparative purposes.

Expanded View Figure Legend
METABOLIC ROUTING MAINTAINS THE UNIQUE FATTY ACID COMPOSITION OF PHOSPHOINOSITIDES (2022)
Figure 2

Figure 2: Metabolic fate of de novo synthesized phosphatidic acid using different fatty acid substrates in microsomal membrane preparations. (A) incorporation of 14C-labeled glycerol 3-phosphate (G3P) into various lipids in the presence of palmitoyl-CoA (10 µM) in microsomal membranes prepared from HEK293-AT1 cells. After incubations at room temperature for 30 min, lipids were extracted and separated by TLC before quantification using autoradiography and phosphorimaging. For more experimental details, refer to the Materials and Methods. Lipid species corresponding to the major spots visualized are indicated. Note the appearance of phosphatidylcholine (PC) and cytidine diphosphate-diacylglycerol (CDP-DAG) in the presence of cytidine trisphosphate (CTP), as well as the production of phosphatidylinositol (PI) only when both CTP and myo-inositol are present. Also, there is a relatively small effect of the DAG kinase inhibitor (DGKi) on CDP-DAG and PI formation in spite of the fact that plenty of phosphatidic acid (PA) is available from de novo synthesis (although it should be noted that no ATP was added during these reactions). A representative experiment is shown, but these results have been reproduced in other replicates using different treatment combinations. The insert shows the effect of the DGK inhibitor on PI labeling. (B) Effect of lipin-1γ overexpression on the metabolic fate of de novo synthesized PA. Experiments were performed as in panel A, except that the membranes were also prepared from cells expressing a GFP-tagged lipin 1γ, as indicated. Note the large amount of TG and increased production of PC under these conditions. Results of a representative experiment are shown, and these experiments were repeated with identical results. (C) Effect of different fatty-acyl-CoA analogs (each used in 10 µM) on the metabolic fate of de novo synthesized PA. Note the preferential conversion of oleoyl-CoA containing PA into TG and the very poor incorporation of stearoyl-CoA into TG. Arachidonoyl-CoA is a relatively poor substrate for the generation of de novo synthesized PA; however, this substrate has the highest conversion rate into PI, which was calculated as the ratio of the PI and PA mass shown in panel D. A representative autoradiogram is shown in (C), while quantified values (means ± range) from two such experiments are shown in (D) and (E).
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling (2020)
Figure 1

Figure 1. The level of LRP6 was reduced in nutrient starvation via endocytosis-mediated lysosomal-dependent degradation.

A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.

B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.

C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.

D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.

E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.

F and G. Loss of LRP6 by serum starvation was mediated in a clathrin or caveolin-1-dependent manner. HEK293 cells were transfected with clathrin (F) or caveolin (G) siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.

H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling (2020)
Figure 3

Figure 3. Regulation of YAP phosphorylation and activity by LRP6 requires Merlin and LATS1/2.

A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.

B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.

C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.

D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis.

E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.

F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM.

G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.

H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling (2020)
Figure 4

Figure 4. Merlin changed binding partners from LRP6 to LATS under serum starvation.

A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.

B-C. The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 (B) or anti-Merlin (C) antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.

D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.

E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.

F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates.

G. Schematic summary of Figure 4 experimental results.
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling (2020)
Figure 5

Figure 5. O-GlcNAcylation of LRP6 was regulated by nutrient status and modulation of global O-GlcNAcylation affected the interaction between Merlin and LATS.

A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates.

B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.

C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.

D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.

E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.

F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.

G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.