EMBO/soda-nel huggingface dataset:
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 6

Figure 6: Model of the Cvt pathway.

(A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide.

(C) These assemblies are capable of binding Atg19 that forms a trimer in solution.

(D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site.

(E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol.

(F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article.

(H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization.

(I) Ams1 does not undergo a maturation process in the vacuole.
PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522 (2021)
Figure 2

Figure 2. Receptor-mediated release of Ca2+ in macrophages and microglia.

A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001).

B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001).

C. PLCG2R522 hIPSC-derived microglia show increased cytosolic Ca2+ influx in comparison to controls following activation of PLCγ2 using anti-CD32. Cytoplasmic Ca2+ increase following activation of PLCγ2 using anti-CD32. Three independent PLCG2R522CRISPR-engineered clones were examined. Data shown is mean±SD of 4 independent experiments and were analysed by one-way ANOVA with Tukey's multiple comparison test (* = p<0.05; ** = p<0.01).
PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522 (2021)
Figure 6

Figure 6. Protective R522 M-MOP and microglia display increased depletion of PIP2 after exposure to physiologically-relevant stimuli compared to P522 M-MOP and microglia.

A-D.The level of PIP2 in M-MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1-42 (A). The levels of PIP2 was also measured using a plate reader after exposure to physiologically relevant stimuli in M-MOPS and primary mouse microglia (B). DAG level were measured using a live cell assay in M-MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522). See also Appendix Figure S2.
PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522 (2021)
Figure 7

Figure 7: Protective R522 M-MOP and microglia display increased depletion of PI(4,5)P2 without corresponding compensation from other PIP species

A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P2 (A), PI(3,4)P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)
PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522 (2021)
Figure 8

Figure 8 In vivo basal PIP2 levels are decreased in microglia of Plcg2R522 mice compared to Plcg2P522 mice.

A-C. Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001. See also Appendix Figure S7.
Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner (2013)
figf4

(a) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8–9 technical replicates from two independent biological aging replicates for all data, F = 7.52, one-way-ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9a. (b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b. (c–f) Adult brains of 3-d-old and 30-d-old Spd− flies, as well as 3-d-old and 30-d-old Spd5mM+ flies immunostained for ref(2)P. Scale bar represents 25 μm. (g) Quantification of ref(2)P intensity in the central brain region normalized to 3-d-old Spd− (n = 12–13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.
Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner (2013)
figf5

(a) Aversive associative memory performance 3 min after training (STM) was markedly reduced in 3-d-old Atg7−/− flies (Atg7d14/Atg7d77) compared with Atg7−/+ flies (Atg7d14/CG5335d30) and wild-type flies (Atg7+/+) (n = 8–11 independent experiments, F = 25.82, one-way ANOVA with Bonferroni correction). The CG5335d30 line harbors an Atg7+ chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control. (b) Olfactory learning was disrupted in 3-d-old female flies homozygous for Atg8a hypomorph (Atg8a−/− or Atg8aEP362/Atg8aEP362) (n = 8–9 independent experiments, F = 22.8, one-way ANOVA with Bonferroni correction). (c) STM is severely impaired in 20-d-old Atg7−/− (Atg7d14/Atg7d77) flies and 20-d-old Atg7−/+ heterozygous (Atg7d14/CG5335d30) when compared to age-matched wild-type flies (n = 8–9 independent experiments, F = 18.02, one-way ANOVA with Bonferroni correction). (d) Similarly, STM was markedly decreased in 20-d-old Atg8a−/− (Atg8aEP362/Atg8aEP362) mutant female flies compared with control flies (Atg8a+/+) without any effect by spermidine feeding (n = 8–9 independent experiments, F = 68.43, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m. The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5.
Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner (2013)
figf7

(a) Aversive associative memory performance in 3 min after training (STM) in 3-d-old female flies with pan-neuronal expression of UAS–Odc-1 (n = 7 independent experiments, F = 0.04, one-way ANOVA with Bonferroni correction). (b) Aversive associative memory performance in 3 h after training (ITM), ARM and ASM of 3-d-old appl-driven UAS–Odc-1 female flies compared with control flies (n = 7 independent experiments; F = 1.95 for ITM, F = 0.10 for ARM, F = 1.05 for ASM; one-way ANOVA with Bonferroni correction). (c) Pan-neuronal expression of the UAS–Odc-1 in a wild-type background suppressed AMI in 30-d-old female flies (n = 7 independent experiments, F = 21.35, one-way-ANOVA with Bonferroni correction). (d) ITM, ARM and ASM of 30-d-old female flies with pan-neuronal expression of UAS–Odc-1 compared with their genetic controls (n = 7–8 independent experiments; F = 17.49 for ITM, F = 1.58 for ARM, F = 24.61 for ASM; one-way ANOVA with Bonferroni correction). (e) Expressing Odc-1 in just the mushroom body was sufficient to protect from STM decline in 30-d-old flies (n = 9–12 independent experiments, F = 9.25, one-way ANOVA with Bonferroni correction). (f) ITM, ARM and ASM of 30-d-old flies expressing mushroom body–specific Odc-1 compared with their genetic controls (n = 8–9 independent experiments; F = 11.73 for ITM, F = 0.50 for ARM, F = 16.6 for ASM; one-way ANOVA with Bonferroni correction). **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.
Deficiency in Intestinal Epithelial O-GlcNAcylation Predisposes to Gut Inflammation (2018)
Figure 1

Figure 1 - Defective O-GlcNAc signaling in intestinal epithelial cells in IBD patients.

A, B Representative images of OGT (A) and O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125)

C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008)

D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16).

Data information: Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 by one-way ANOVA with the Dunnett post hoc test.
Deficiency in Intestinal Epithelial O-GlcNAcylation Predisposes to Gut Inflammation (2018)
Figure 2

Figure 2 - Loss of OGT in IECs causes intestinal damages in mice.

A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015).

B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001).

C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice.

D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001).

E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439).

F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm.

Data information: Data are represented as mean ± SEM. *** P < 0.001 by two-tailed t-test (A), Mantel-Cox test (B and E), or one-way ANOVA with the Dunnett post hoc test (D).
Deficiency in Intestinal Epithelial O-GlcNAcylation Predisposes to Gut Inflammation (2018)
Figure 6

Figure 6 - Paneth cell dysfunction in OGT-deficient mice.

A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048).

B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm.

C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031).

D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice.

E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells.

F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm.

G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462).

H Representative images of cultured organoid on Day 3.

I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5).

J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J), colitis scores (K), and RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049)

Data information: Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01 by two-tailed t-test.
Deficiency in Intestinal Epithelial O-GlcNAcylation Predisposes to Gut Inflammation (2018)
Figure 7

Figure 7 - O-GlcNAc regulation of STAT1 in IECs.

A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice.

B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown.

C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots.

D Most significantly enriched canonical signaling pathways in differentially expressed genes, determined by the Comparison Analysis module in the IPA program.

E Top candidate transcription factors controlling the observed differentially expressed genes, predicted by DiRE.

F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting.

G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylation (G) and STAT1 O-GlcNAcylation (H) were determined.

I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later.

J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003).

Data information: Data are represented as mean ± SEM. *** P < 0.001 by two-way ANOVA followed by Tukey"s multiple comparisons test.
Deficiency in Intestinal Epithelial O-GlcNAcylation Predisposes to Gut Inflammation (2018)
Figure 8

Figure 8 - Elevation of protein O-GlcNAcylation diminishes DSS-induced intestinal inflammation.

A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445)

C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117).

D-G Albumin levels in feces (D), colon length (E), H&E staining of colon tissues (F) and RT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301)

Data information: The data are represented as mean ± SEM. *, P < 0.05 by two-way ANOVA followed by Bonferroni corrections (B) and two-tailed t-test (C, E and G).

Expanded View Figure Legends
Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for g-secretase activity (2022)
Figure 3

Figure 3: Endogenous Fn14 is processed by protease γ-secretase

A. Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14.

B. Quantification of blots from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1.

C. Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14.

D. Quantification of blot from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1.

E. sFn14 is reduced upon γ-secretase inhibition in MDA-MB-231 cells. Conditioned media of the treated cells were collected after overnight DAPT (1 μM) or vehicle treatment. sFn14 concentration was measured by human Fn14 ELISA.

F. sFn14 is reduced upon γ-secretase inhibition in SKOV-3 cells. Conditioned media of the treated cells were collected after 48 h DAPT (1 μM) or vehicle treatment. sFn14 concentration was measured by human Fn14 ELISA.

Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B and D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. For panels E and F, two-tailed unpaired t-test is used. The p-values that are above 0.05 have not been included into the panels.
Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for g-secretase activity (2022)
Figure 4

Figure 4: Surface level of endogenous Fn14 is increased upon γ-secretase inhibition

A. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with γ-secretase inhibitor DAPT (1 μM) or vehicle overnight. The lysate was collected and blotted against Fn14 (C-terminal antibody) or calnexin as loading control. Shown are representative blots from N=4 experiments.

B. Quantification of blot in panel A. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments.

C. MDA-MB-231 cells were transfected and treated as in panel A. The treated cells were suspended and labeled with ITEM-4 antibody that targets an extracellular site of Fn14, or isotype control. Shown are representative histograms from N=3 experiments.

D. The mean intensity of the measurement from panel C. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=3 experiments.

Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels. Number of biological replicates performed is indicated in the corresponding panel legend.
Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for g-secretase activity (2022)
Figure 6

Figure 6: γ-Secretase mediates proteolysis of Fn14 in primary cells from human glioblastoma biopsy samples

A. Cellular Fn14 in ex vivo glioblastoma samples increased upon γ-secretase inhibition. Primary cells from four different glioblastomas were treated with DAPT (1 μM) or vehicle overnight. Lysates were blotted against Fn14 and calnexin as loading control.

B. Quantification of the blot in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14 vehicle condition was used for normalization.

C. sFn14 was strongly reduced upon γ-secretase inhibition in primary GBM cells. Conditioned media of the treated cells from panel A were collected after overnight DAPT (1 μM) or vehicle treatment. sFn14 levels in these samples were measured by human Fn14 ELISA.

Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.
Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for g-secretase activity (2022)
Figure 7

Figure 7: In vivo sFn14 is generated by γ-secretase activity

A. Mice were treated with single dose of 100 mg/kg DAPT (or vehicle DMSO) and their plasma was collected at the indicated timepoints after dosing. Individual mice were used at each time point. sFn14 levels in these samples are displayed (N=4).

B. Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM (iGBM, 40 patients) or a recurrent GBM (rGBM, 20 patients) were collected and sFn14 levels were measured by ELISA.

C. Plasma samples from 10 ovarian cancer patients with low/middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA. The tumor burden was clinically determined by computer tomographic scans.

D. Serum samples from 10 patients with refractory multiple myeloma were collected pre- and post-treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10).

Data information: For panel A, data is shown as mean ± SEM. Different mice were used for each time point. Two-tailed unpaired t-test was applied. For panel B and C, median of the measurements, along with interquartile range borders are displayed. In panel B, ordinary one-way ANOVA with Tukey's multiple comparison test was applied, but no value passed our p-value display threshold of 0.05.

For panel C, a two-tailed unpaired t-test was applied, but no value passed our p-value display threshold of 0.05. For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment. For all the panels, the number of biological replicates is reported in the corresponding panel legend.