EMBO/soda-nel huggingface dataset:
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 4

Figure 4. TRAF3IP3 mediates TRAF3 recruitment to MAVS upon virus infection.

A pcDNA-FLAG-TRADD and pcDNA-FLAG-TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively. ISG54 and IL6 induction were measured thirty-six hours post-transfection by qPCR. Expression levels were shown in Fig EV4B. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 NS indicates no statistically significant difference.

B HEK293T cells were transfected with pcDNA3-HA-TRAF3IP3, in combination with pcDNA3-FLAG- RIG-I, MAVS, TBK1, IRF3, TRAF2, TRAF3, TRAF5, or TRAF6 as indicated. Thirty-six hours after transfection, cells were collected and anti-HA immunoprecipitations were performed. IP products were subjected to immunoblotting. Protein expression levels were shown in Fig EV4C.

C shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting.

D Wild type and Traf3ip3-/- HEK293T cells were transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours post-transfection, cells were infected with or without VSV. Twelve hours post-infection, cells were collected and subjected to immunoprecipitation assay and immunoblotting.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 5

Figure 5. TRAF3IP3 is accumulated on the mitochondria after virus infection.

A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions, i.e., cytoplasm, membrane and nuclei extracts, which were then subjected to Immunoblotting analysis as indicated.

B Membranous fraction obtained in Fig 5A were further separated into mitochondria, Golgi and ER extracts as described in methods, followed by immunoblotting analysis.

C-E Immunofluorescent microscopic imaging for TRAF3IP3. HeLa cells were transfected with pcDNA3-FLAG-TRAF3IP3. Twenty-four hours after transfection, cells were infected with or without SeV for twelve hours. Anti-FLAG M2 (FITC) was used for immunofluorescence staining of FLAG-TRAF3IP3. Nuclei were stained with DAPI. Mitochondria was stained with Mitotracker Red (C). Calnexin (an ER protein) was stained for ER (D). GM130 (a Golgi protein) was stained for Golgi (E). Scale bar represents 5 micrometers.

F Quantitative analysis of yellow color as shown in Fig 5C, D and E, indicating TRAF3IP3 localization on various organelles with or without virus infection. The quantification was performed with ImageJ. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. ***P<0.001. NS indicates no statistically significant difference.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 6

Figure 6. TRAF3IP3 mediates interferon response to virus infection in BMDMs and PEMs from mice.

A-E BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.

F BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were infected with or without VSV for six hours. Cells were then collected and following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation.
TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity (2019)
Figure 7

Figure 7. Traf3ip3-deficiency attenuates antiviral immunity in mice.

A-F Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were measured respectively by qPCR. VSV RNA levels were measured by qPCR (E). The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.

G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05
Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration (2020)
Figure 1

Figure 1. lid-1(xd288) and atgl-1(xd314) display ectopic LD accumulation in neurons.

(A) lid-1(xd288) and atgl-1(xd314) mutants show LD accumulation in most neurons in C. elegans, including neurons in the head region, tail and ventral nerve cord. xdIs109 is a stable transgenic line with pan-neuronal expression of the LD marker PLIN1::GFP. The white arrows indicate PLIN1::GFP-positive LDs in the ventral nerve cord of mutants. Scale bar: 20 μm. The right panels depict the head region. Scale bar: 5 μm. The insets in the right panels are enlarged views of PLIN1::GFP rings, representing LDs.

(B) Quantification of the numbers of LDs in head region neurons (each dot represents one worm). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not statistically significant. Data show mean ± SEM. n ≥ 9.

(C-D) The genetic loci of lid-1 and atgl-1. The coding regions are in light blue boxes and the noncoding regions are shown as lines. The UTRs are in red boxes. xd288 is a point mutant of lid-1 and causes a missense S111L mutation in the α/β hydrolase domain. xd314 and xd310 are point mutants of atgl-1. xd314 harbors a missense G19R mutation and xd310 harbors a missense G210E mutation.

(E) Oil red O staining show dramatically increased neutral lipids in the head region of lid-1(xd288) and atgl-1(xd314) compared with N2. Scale bar: 10 μm.

(F-H) EM images of neurons. The red dashed line marks the outline of neurons. The red arrows mark LDs. Scale bar: 0.5 μm. The scale bar in enlarged images is 100 nm.

(I) Serial sections of atgl-1(xd314) show two LDs (indicated by blue and red arrows) in a neuron cell body. Scale bar: 0.5 μm.
Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration (2020)
Figure 3

Figure 3. atgl-1 autonomously regulates neuronal LD dynamics.

(A) The expression pattern of atgl-1 viewed by Patgl-1::GFP transcriptional fusion reporter. Patgl-1::GFP expression is high in intestine. Relatively low fluorescent signals are found in nerve ring, motor neuron commissure, ventral nerve cord and tail. Scale bar: 20 μm.

(B) atgl-1 is expressed in neurons. There is no fluorescent signal when Patgl-1::GFP1-10 and Prab-3::GFP11 are expressed separately. When Patgl-1::GFP1-10 and Prab-3::GFP11 are co-expressed, the GFP signals are found in the head region, tail and ventral nerve cord, where most neuronal cell bodies are located. The white lines mark the outline of worms. Scale bar: 50 μm.

(C) atgl-1 is expressed widely in neurons. The left panels depict the whole worm. Scale bar: 50 μm. The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels. The signals of mCherry and GFP merge very well. Scale bar: 20 μm.

(D) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds. The accumulation of LDs in head neurons of atgl-1(xd314) mutants can be rescued by expressing wild-type atgl-1 in neurons (Punc-119::atgl-1), but not in intestine (Pvha-6::atgl-1), glia (Phlh-17::atgl-1) or hypodermis (Pajm-1::atgl-1). Scale bar: 5 μm.

(E) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 7.

(F) Neuron-specific knockout of atgl-1 causes LD accumulation in neurons, similar to atgl-1(xd314) mutants. Scale bar: 5 μm.

(G) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. t-test was used between the groups under the crossbars. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 9
Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration (2020)
Figure 4

Figure 4. atgl-1(xd314) and lid-1(xd288) show a gentle touch sensation defect.

(A) LDs accumulate in touch neurons in atgl-1(xd314) visualized by SRS (Stimulated Raman Scattering). The zdIs5 reporter marks touch neurons. The white arrows indicate LDs in touch neurons in atgl-1(xd314). Scale bar: 10 μm.

(B) atgl-1(xd314) and lid-1(xd288) show a gentle touch sensation defect, which is rescued by the neuron-specific expression of atgl-1 and lid-1, respectively. The fat-4 mutation enhances the touch sensation defect in atgl-1(xd314) and lid-1(xd288). Wild type and mec-4(u253) act as the negative control and positive control, respectively. The data were analyzed using Kruskal-Wallis test with Dunn's test. Asterisks denote significant differences as compared to wild type. ns: not statistically significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. # signify significant differences between the groups under the crossbars. ##P < 0.01. Data show mean ± SEM. Number of worms analyzed for each strain n =25.

(C) The pathway of PUFA synthesis in C. elegans. LA: linoleic acid. ALA: alpha-linolenic acid. GLA: gamma-linoleic acid. STA: stearidonic acid. DGLA: dihomo-gamma-linoleic acid. ETA: eicosatetraenoic acid. AA: arachidonic acid. EPA: eicosapentaenoic acid. Fatty acids are also indicated by chemical abbreviations. For example, the abbreviation C18:2n-6 means 18 carbons with two double bonds and the first double bond is located at ω-6.
Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration (2020)
Figure 5

Figure 5. PUFA synthesis defect significantly enhances the beneficial effect of atgl-1 mutants in preventing the neurodegeneration of mec-4(d).

(A) Image of touch neurons viewed by zdIs5 in different genetic backgrounds. Scale bar: 20 μm.

(B) Quantification of the percentage of animals with 0, 1, 2, 3, 4, 5 and 6 surviving touch neurons in different genetic backgrounds. Data show mean ± SEM. At least 50 animals per strain were analyzed in each experiment. Number of experiments n = 5.

(C) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in (B). The lid-1(xd288) and atgl-1(xd314) mutations significantly increase the number of surviving neurons. Neuron-specific expression of atgl-1 or lid-1 reverses the protective effect of atgl-1 or lid-1 mutation. The data were analyzed using one-way ANOVA with Turkey's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n = 5, with at least 50 animals per strain analyzed in each experiment.

(D) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. Mutations of fat-1, fat-3 and fat-4 significantly enhance the suppression effect of atgl-1(xd314) in preventing the neurodegeneration of mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. ### and #### denote significant differences as compared to atgl-1(xd314); mec-4(d). Asterisks signify significant differences as compared to mec-4(d). ns: not statistically significant. ****P < 0.0001. ###P < 0.001, ###P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.

(E) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids. AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ns: not statistically significant. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.
Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration (2020)
Figure 6

Figure 6. PUFA-containing phospholipids regulate neurodegeneration of mec-4(d).

(A) Lipidomic data show that the relative content of total C20:3 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation.

(B) Lipidomic data show that the relative content of total C20:4 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation.

(C) Lipidomic data show that the relative content of total C20:5 is slightly increased in atgl-1(xd314); mec-4(d) but is dramatically decreased in fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d), which matches the role of FAT-4 in synthesizing C20:5 (EPA).

(D) The percentage of C20:3-, C20:4- or C20:5-containing TAGs to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing TAGs to total lipids containing C20:4 is significantly increased in atgl-1 and fat-4 single mutants. The percentage is further increased in atgl-1 and fat-4 double mutant.

(E) The percentage of C20:3-, C20:4- or C20:5-containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing phospholipids to total lipids containing C20:4 is significantly decreased in atgl-1 and fat-4 single mutants. The percentage is further decreased in atgl-1 and fat-4 double mutant.

(F) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. mboa-7 mutation significantly increases the percentage of three or more surviving touch neurons in mec-4(d), atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. **P < 0.01, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 4, with at least 50 animals per strain analyzed in each experiment.

(G) The percentage of C20:4-containing phospholipids to total lipids that have C20:4 is dramatically decreased in mboa-7(gk399); atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks signify significant differences as compared to mec-4(d). ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.

Data information: (A-E) FFA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 1

Figure 1. MDMX inhibits CK1α kinase activity.

(A) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding.

(B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.

(C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table. See Fig EV2A-B for same data fitted to competitive inhibition and allosteric sigmoidal models.

(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. See Fig EV2C for same data fitted to mixed inhibition model.
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 2

Figure 2. MDMX expression inhibits CK1α activity in cells.

(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot.

(C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot.

(D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers.

(E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay. (F) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 3

Figure 3. Endogenous MDMX expression inhibits CK1α activity.

(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot.

(E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 4

Figure 4. The p53-binding and RING domains regulate MDMX ability to inhibit CK1α.

(A) Diagram of MDMX mutant constructs and summary of CK1α inhibitory efficiency. The result of MDMXSG was based on Figure 1B (position of W200S/W201G mutation is marked with **).

(B, C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 5

Figure 5. P53 cooperates with MDMX to inhibit CK1α.

(A) Diagram of MDMX intramolecular interactions and communication between p53 and CK1α binding.

(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.

(C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot.

(D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot.

(E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 6

Figure 6. Tumor-derived CK1α mutants are hyper-sensitive to inhibition by MDMX.

(A) H1299 cells were transfected with MDMX and CK1α mutants. MDMX-CK1α binding was determined by IP-western blot.

(B) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.

(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.

(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 7

Figure 7. Disruption of MDMX-CK1α interaction in vivo down regulates Wnt pathway.

(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.

(B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test).

(C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines.

(D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines.

(E) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test).

(F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).
MDMX inhibits Casein Kinase 1α activity and stimulates Wnt signaling (2020)
Figure 8

Figure 8. Disruption of MDMX-CK1α binding attenuates cell proliferation.

(A) Identical starting number of cells from the MEF lines were serially passaged following identical schedules. The differences in cell number were determined after 3 and 5 passages. The results are average of 3 experiments (mean +/- SD).

(B) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days. Colony formation efficiency was determined by crystal violate staining.

(C) Survival curves of p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt mice. Thymic lymphomas were the major cause of mortality in both genotypes.

(D) A model of Wnt signaling regulation by MDMX. In the absence of stress, MDMX inhibits CK1α, increases basal expression of Wnt/β-catenin target genes and stimulates proliferation. Interaction with mutant p53 stimulates MDMX-CK1α binding and promotes proliferation. DNA damage promotes MDMX degradation and inhibits MDMX-CK1α binding, reducing Wnt signaling.

Expanded view figure legend