EMBO/soda-nel huggingface dataset:
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 6

Figure 6. ABRO1 facilitates NLRP3 deubiquitination dependent on BRCC3.

A WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.

B Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.

C Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.

D A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.

E-H Abro1+/+, Abro1−/−, Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody (E, F). CBA analysis of IL-1β in culture supernatants (G, H).

Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (G and H) or are representative of three independent experiments (A-F). *P < 0.05, **P < 0.01; ns, not significant; two-tailed unpaired t-test (G and H).

Source data are available online for this figure.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 7

Figure 7. BRCC3 mediates activation of the NLRP3 inflammasome in vitro and in vivo.

A Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.

B Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).

C Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.

D LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.

E Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.

F ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.

G, H Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G) and flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.

Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (A and C) or are representative of three independent experiments (B and D). In (E-H), data are pooled from two (all PBS groups) or three (E, LPS groups of F, MSU groups of G-H) independent experiments. In (F-H), data are shown in box-and-whisker plots: dots indicate individual mice; Box-plot center line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. *** P < 0.001; log-rank test (E) or two-tailed unpaired t-test (A, C, F- H).

Source data are available online for this figure.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 1

Figure 1. HP1c is involved in Notch-dependent Drosophila notum bristle and wing development.

(A) Workflow of the network-based analysis in silico and functional screen in vivo.

(B) The number of high-risk genes (DIOPT score≥5) in colorectal cancer and gastric cancer from genome-wide prediction (total: 17,903 genes).

(C-G) Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F) and mutant (G) flies also exhibit supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.

(H-M) Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 2

Figure 2. HP1c, as well as Notch signaling, regulates Drosophila gut homeostasis.

(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.

(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 3

Figure 3. HP1c genetically represses the Notch signaling pathway.

(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.

(E-J) Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 4

Figure 4. HP1c represses Notch target genes at the transcriptional level.

ChIP-qPCR results show the association of HP1c with Notch target genes compared to RFP control. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

(B) RT-qPCR analysis determined the expression of Notch target genes when HP1c was depleted (n=3 technical replicates,, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, ***p < 0.001).

(C) Immunostaining result of Drosophila wing disc when control or HP1c was KD by en-Gal4, which knocked down target gene in RFP marked region, and HP1c KD caused upregulation (arrow) and expansion (arrowhead) of GFP signal under the control of a Notch response element (NRE) that the GFP fluorescence intensity represents the activity of Notch signaling. Scale bars, 20 μm.

(D) Quantification of results in © showing that the relative GFP intensity in HP1c KD wing discs is about threefold higher than that in control (n=5 biological replicates, mean±s.d.). Data are evaluated with Student's t-test (**p < 0.01).

(E, F) NRE-GFP signals of S2 cells carrying NICD OE, Hairless KD, Su(H) KD, dSir2 KD and HP1c KD (E), and the statistical results (F) (n=4 biological replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 200 μm.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 5

Figure 5. HP1c interacts with Su(H), the core transcription factor of Notch signaling.

(A) Yeast two-hybrid analysis identified that Su(H) is the only Notch signaling key component that interacts with HP1c, while lamin mated with T-antigen was used as negative control and p53 mated with T-antigen as positive control. The interactions between HP1c and Su(H) were confirmed in both orientations. (BD: binding domain, AD: activation domain, QDO/X/A: Quadruple dropout medium: SD/-Ade/-His/-Leu/-Trp supplemented with X-a-Gal and Aureobasidin A).

(B) The main functional domains of HP1c and Su(H).

(C) The CSD domain of HP1c and the BTD domain of Su(H) are responsible for the interaction by yeast two-hybrid analysis.

(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.

(F) Plasmids expressing HP1c CSD domain and Su(H) BTD domain were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.
HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) (2021)
Figure 6

Figure 6. HP1c associates with Notch target genes in a Su(H)-dependent manner.

(A) Confocal images of the polytene chromatin show strong co-localization of HP1c and Su(H), with enlarged images are also shown. Arrowheads in (A''') indicate HP1c (red) and Su(H) (green) signals, while yellow arrowheads indicate co-localizations. Scale bars, 20 μm.

(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).

(C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).

(D-E), ChIP-qPCR results show the association of Flag-tagged HP1c and chromo-domain depleted HP1c mutant (HP1c △CD) with Notch target genes. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). HA antibody is used as a control. Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. (2010)
Figure 1.

Involvement of Atg14L in the initial phase of autophagosome formation. (a) HEK293 cells stably expressing GFP-DFCP1 were transfected with or without shRNA against Atg14L. The cell lysates were subjected to immunoblotting with the indicated antibodies. (b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation. (d) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods. Equivalent amounts of total, cytosolic, and ER fractions were subjected to immunoblotting with each organelle marker antibody. The percentage of recovery in each fraction is presented as mean ± SD values. (e) HEK293 cells stably expressing GFP-DFCP1 were transiently transformed with Flag-tagged Atg14L and subjected to starvation. The cells were fixed and stained with the indicated antibodies. Insets show higher magnification of the boxed areas. (f) HEK293 cells were transfected with GFP-Atg14L with or without mCherry-linked dominant-negative ULK1, and these cells were cultured in complete or starvation medium for 60 min. Bars, 10 µm.
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. (2010)
Figure 2.

Domain analysis of Atg14L structure. (a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)–tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining. (b) Diagram of Atg14L deletion mutant construction. (c) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti-GFP antibody. Each precipitant was subjected to immunoblotting with the indicated antibodies. IP, immunoprecipitation. (d) Alignment of N-terminal conserved region of Atg14 orthologues. Conserved cysteine repeats are boxed. The degree of sequence conservation is denoted: asterisks indicate a perfect match among interspecies, periods indicate mostly conserved, and colons indicate more conserved than the periods. Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr), Drosophila melanogaster (Dm), Candida glabrata (Cg), and Saccharomyces cerevisiae (Sc) are shown. (e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. (2010)
Figure 3.

Ectopic expression of Atg14L in the plasma membrane. (a) A549 cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14LCAAX and subjected to starvation. The cells were fixed and stained with anti–Beclin-1 antibody. Higher magnification of the boxed areas is shown below. (b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. (2010)
Figure 4.

Autophagy induction by overexpressed Atg14L. A549 cells were transiently transfected with adenovirus harboring GFP-Atg14L or GFP-Atg14L4C4A. (a and b) The cells before starvation (a) or after starvation (b) were fixed and stained with anti-LC3 antibody. (c) The number of LC3-positive puncta per cell was counted, and mean ± SD values are presented. Bars, 10 µm.
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. (2010)
Figure 5.

The effect of Atg14L localization on its function. (a) Atg14L knockout mouse ES cells were transfected with GFP, GFP-Atg14L, GFP-Atg14L4C4A, or GFP-ER-Atg14L4C4A of the pCAG vector and subjected to starvation. The cells were fixed and immunostained with anti-Atg16L or anti-LC3 antibodies. (b and c) The number of Atg16L-positive puncta (b) and LC3-positive puncta (c) per cell was counted in at least 10 GFP-positive cells, and mean ± SD values are presented. Bars, 10 µm.
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 1

Figure 1 - Wnt7a increases TNTs and vesicle transfer in CAD cells.

Representative confocal images of CAD cells exposed for 4 h to the indicated treatments. Cells were fixed and then stained with WGA (green) and DAPI (blue).

Percentage of TNT-connected CAD cells with each of the indicated treatments.

Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.

Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.

Top: Schematic of the culture system used to assess vesicle transfer by secretion. Donor cells (labeled with DiD) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Then acceptor cells (labeled with CTG) were treated for 4 h with the conditioned media from donor cells. Bottom: Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.

Percentage of acceptor cells containing DiD-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells.

Data information: In (A, C, E) scale bars represent 10 µm. In (B, D, F) graphs represent the average of 3 independent experiments showing mean ± SEM. Statistical significance was calculated with respect to control (Ctrl); * p ≤ 0.05; ** p ≤ 0.01, NS = not significant (one-way ANOVA).
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 2

Figure 2 - Pharmacological inhibition of CaMKII decreases TNT's formation and function.

Representative confocal images of CAD cells cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Cells were fixed and stained with WGA (green) and DAPI (blue).

Percentage of TNT-connected CAD cells with each of the indicated treatments.

Representative confocal images of co-cultured donor and acceptor CAD cells. Cells were co-cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.

Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.

Representative confocal images of CAD cells stained for vinculin (red) and HCS CellMask™ Blue after being treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell. This area is defined by the board of the HCS CellMask™ Blue staining and the vinculin staining around it.

Average number of vinculin puncta per cell with the indicated treatments.

Data information: In (A, C, E) scale bars represent 10 µm. In (B, D, F) graphs represent the average of 3 independent experiments showing mean ± SEM. Statistical significance was calculated with respect to control (Ctrl); * p ≤ 0.05, ** p ≤ 0.01, NS = not significant (one-way ANOVA).
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 3

Figure 3 - TNT's formation and function depend on βCaMKII phosphorylation status.

Representative confocal images of CAD cells expressing the indicated GFP-fusion proteins. Top: Cells showing WGA (white) and DAPI (blue) staining. Bottom: Cells showing phalloidin (red) and DAPI (blue) staining. Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R, is rather punctate than diffused.

Percentage of TNT-connected CAD cells 24 h after transfection with indicated GFP-fusion proteins.

Percentage of transfection rate of indicated GFP-fusion proteins in CAD cells.

Percentage of acceptor cells containing DiD-labeled vesicles when acceptor cells were co-cultured with donor cells expressing the indicated GFP-fusion proteins.

Top: Schematic of the co-culture system used to assess vesicle transfer in transfected cells. Donor cell were transfected with the indicated GFP-fusion proteins and then labeled with DiD. Acceptor cells were transfected with a H2B-mCherry plasmid. After 24 h, the cells were detached, replated in 1:1 ratio and cultured for 4 h. Cells were then fixed and stained with HCS CellMask™ Blue. Bottom: Representative confocal images of co-cultured donor and acceptor CAD cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.

Data information: In (A, E) scale bars represent 10 µm. In (B, C, D) graphs represent the average of 4 independent experiments showing mean ± SEM. Statistical significance was calculated with respect to control (Ctrl); * p ≤ 0.05, NS = not significant (one-way ANOVA).
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 4

Figure 4 - Actin-binding activity of βCaMKII affects the stability of TNTs.

Time lapses showing a TNT connecting two transfected cells. Top: GFP-vector transfected cells. Middle: GFP-βCaMKII WT transfected cells. Bottom: GFP-βCaMKII T287D transfected cells. Cells were stained with WGA (white). Yellow arrowheads point out a TNT in each condition. Scale bars represent 10 μm.

TNT's average duration between GFP-vector, GFP-βCaMKII WT and GFP-βCaMKII T287D transfected cells. Graph represents the average lifetime of 15-20 TNTs per condition ± SEM. Statistical significance was calculated with respect to control (Ctrl); ** p ≤ 0.01, NS = not significant (one-way ANOVA).

CODIM super-resolution images (3 right side panels) of TNTs shown in the confocal expanded field images (left panels) of transfected cells showing expression of indicated GFP-tagged proteins (green). Cells were stained for F-actin with phalloidin (red). Scale bars in the expanded fields represent 5 µm and 1 µm in the insets.

Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII (green) in CAD cells. Cells were stained with DAPI (blue) and phalloidin (red). Yellow arrows point to βCaMKII-positive puncta inside a TNT. Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection.
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 5

Figure 5 - Wnt7a regulates actin dynamics in CAD cells overexpressing βCaMKII.

Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm.

Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a.

Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.