EMBO/soda-nel huggingface dataset:
Identification of TOR-responsive slow cycling neoblasts in planarians (2020)
Figure 6

Figure 6: Lrig-1 is required for sustained regeneration.

A Representative WISH images for Lrig-1.

B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates).

C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).

D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17).

E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.

F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18).

G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.

Data information: Data in B-G are presented as mean ± s.d. Statistical significance was assessed using Welch's t-tests (p values are indicated; ns, not significant (p > 0.05)). In B and C, data was obtained at 5fd5. In D-G, animals were initially amputated at 5fd5. Second amputations were performed at 7 dpa following the first amputation. Data are representative of 3 (B and C) or 2 (D-G) independent experiments (with the exception of the homeostatic data in D and F, for which a single experiment was performed). In D-G, combined data from both independent experiments is shown.
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 1

<italic>Apc</italic> loss, GSK3 loss and homozygous mutation of β-catenin are sufficient to induce a rapid crypt-progenitor phenotype, but not a single β-catenin mutation

Wild-type mice have small defined crypts in the small intestine with little nuclear β-catenin at the bottom of the crypt. The small intestine of AhCreER
APCfl/fl, AhCreER
Catnblox(ex3)/lox(ex3) (day 5) or AhCreER
Gsk3alphafl/fl
betafl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclear β-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm.

A heterozygous activation of β-catenin (AhCreER
Catnblox(ex3)/+) shows no increase in crypt size or nuclear β-catenin at days 5–10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm.

Culture of small intestinal crypts of WT and VilCreER
Apcfl/fl (or AhCreER
Apcfl/fl) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc-deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm.

At day 5 post-induction, only crypts from AhCreER
Catnblox(ex3)/lox(ex3) but not AhCreER
Catnblox(ex3)/+ survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCreER
Catnblox(ex3)/+ compared to spheres from Catnblox(ex3)/lox(ex3) crypts in the first week of culture. Black scale bar, 50 μm.

Quantification of organoids/spheres at day 10 post-induction (N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 2

Activation of one allele of β-catenin in Lgr5-positive stem cells does not result in colonic lesions

Table shows that only homozygous activation of β-catenin (Lgr5CreER
Catnblox(ex3)/lox(ex3)) or loss of Apc (Lgr5CreER
Apcfl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin (Lgr5CreER
Catnblox(ex3)/+) was mutated (chi-squared test, P < 0.001).

Colonic lesions were scored from H&E by identification of disorganised epithelium. Staining for β-catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions. Scale bar, 100 μm.
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 3

Increased E-cadherin:β-catenin levels in colonic crypts of wild-type mice

Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm.

Western blot of purified crypts from the small intestine and colon (N = 4 mice).

qRT–PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann–Whitney U-test.

Proximity ligation assay for E-cadherin and β-catenin. For each mouse (N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann–Whitney U-test; white scale bar, 50 μm.
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 4

E-cadherin saturates with mutant β-catenin over time

Immunoprecipitation (IP) of E-cadherin from VilCreER
Catnblox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.

IP of E-cadherin from VilCreER
Catnblox(ex3)/+ mice at day 4 and day 8. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 5

Haploinsufficiency for E-cadherin in the presence of single allele β-catenin mutation leads to Wnt deregulation

AhCreERCatnblox(ex3)/+ compared to AhCreER
Catnblox(ex3)/+
Cdh1fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCreER
Catnblox(ex3)/+
Cdh1fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU).

Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCreER
Cdh1fl/+, AhCreER
Catnblox(ex3)/+ and AhCreER
Catnblox(ex3)/+
Cdh1fl/+ mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCreER
Catnblox(ex3)/+
Cdh1fl/+ mice (P = 0.028, one-sided Mann–Whitney U-test).

In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreER
Catnblox(ex3)/+
AhCreER
Catnblox(ex3)/+
Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann–Whitney U-test.

Survival of Lgr5CreER
Catnblox(ex3)/+ and Lgr5CreER
Catnblox(ex3)/+
Cdh1fl/+ shows significant acceleration (P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-catenin IHC in contrast to Lgr5CreER
Catnblox(ex3)/+ mice.
E-cadherin can limit the transforming properties of activating β-catenin mutations. (2015)
Figure 6

Human cancers, characterised by β-catenin mutation, are associated with reduction in E-cadherin levels

Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann–Whitney U-test, P = 0.0009051. White scale bar, 50 μm.

Correlation of several Wnt target genes with the expression of E-cadherin (CDH1) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.
The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice (2020)
Figure 1.

Weight loss, virus replication and specific IgG production in hACE2 mice post infection with SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001). To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b). Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001), and the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d). The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).
The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice (2020)
Figure 2.

Gross pathology, histopathology, and immunohistochemistry of the lungs in SARS-CoV-2-infected hACE2 mice.a. Gross pathology and histopathology of lungs from WT-HB-01 mice (3 dpi), ACE2-Mock mice (3 dpi) and ACE2-HB-01 mice (3 dpi and 5 dpi). Postmortem examinations showed focal dark red lesions (red arrow) throughout the dorsal of the right middle lobe of the lung at 3dpi. The lesions progressed into multifocally scattered-dark reddish purple areas and palpable nodules (red arrow) throughout the right lobe of the lung at 5 dpi. Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa (black arrows) and infiltration of lymphocytes (red arrows). The swollen and degenerative alveolar macrophages (green arrows) are scattered within the alveolar cavities at 3dpi and 5 dpi. b. Immunohistochemical examination of lungs of each group. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed in the alveolar macrophage (green arrows), the alveolar epithelia (blue arrows), and the degenerative and being desquamated bronchial epithelial cells (black arrows). Black bar = 100 µm, red bar = 50 µm.
The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice (2020)
Figure 3.

Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 1

Figure 1. TRIM21 directly binds to SIRT5 and promotes its degradation.

(A) Mass spectrometry (MS) assay of indicated electrophoretic bands in Coomassie blue staining (left panel), and proteins pulled down by Flag-tagged SIRT5 from 293T cells were indicated on the right.

(B) Lysates from 293T cells transfected with HA-TRIM21 and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB).

(C, D) Lysates from BMDMs were immunoprecipitated with anti-SIRT5 (C), anti-TRIM21 (D) or control IgG antibodies as indicated and then analyzed by western blotting.(E) Bacterially expressed and purified Flag-SIRT5 were incubated with purified HA-TRIM21 proteins prior to pull-down with anti-HA antibody followed by western blot analysis.

(F) Immunoblot (IB) analysis of whole-cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with Flag-SIRT5 together with HA-TRIM21 or indicated HA-tagged mutant TRIM21 constructs. Cells were treated with 10 μM MG132 for 4 hours before harvesting. HA, hemagglutinin.

(G) IB analysis of WCLs derived from 293T cells transfected with increasing amounts of Flag-TRIM21 construct. Where indicated, 10 μM MG132 was added for 4 hours before harvesting.

(H) 293T cells transfected with control or TRIM21 siRNA for 48 hours were then treated with 100 ng/mL CHX for the indicated time period before harvesting. Protein expression was analyzed by western blotting (left panel). The SIRT5 protein abundance was quantified by ImageJ and plotted as indicated (right panel).

(I) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/ml CHX for the indicated time period before harvesting (left panel). The SIRT5 protein abundance was quantified (right panel).

(J) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for the indicated time period before harvesting (top panel). The SIRT5 protein abundance was quantified (bottom panel).

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 2

Figure 2. TRIM21 is an E3 ligase of SIRT5 promoting its ubiquitination, which is enhanced by LPS challenge.

(A) Immunoblot analysis of WCLs and anti-GST immunoprecipitates derived from 293T cells transfected with the indicated plasmids for 48 hours, cells were then treated with the proteasome inhibitor MG132 for another 4 hours prior to harvesting.

(B) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting. 10 μM MG132 was added for 4 hours before harvesting.

(C) TRIM21 ubiquitinates SIRT5 in vitro. Immunopurified SIRT5 were incubated with purified recombinant TRIM21 or mutant TRIM21 proteins, E1 and E2 (UBE2D2), and ubiquitin as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

(D) Purified SIRT5 protein were incubated with purified recombinant TRIM21, E1 and E2 (UBE2D2), and ubiquitin in the presence or absence of control mIgG or GST proteins as indicated for 90 min at 30℃. SIRT5 enzymatic activity was measured. Data are from n = 3 biological replicates. Data are the mean ± SD. Statistical significance was determined by unpaired two-tailed Student's t-tests.

(E) Purified SIRT5 and mutant SIRT5-10KR proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and/or purified recombinant TRIM21 or control mIgG or GST proteins as indicated at 30℃ for 90 min. SIRT5 ubiquitination and TRIM21 protein levels were analyzed by western blotting with the indicated antibodies.

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 3

Figure 3. TRIM21 catalyzes Lys48-linked ubiquitination of SIRT5.

(A) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

(B) IB analysis of WCLs and anti-HA immunoprecipitates of 293T cells transfected with the indicated plasmids. 48 hours posttransfection, cells were treated with 10 μM MG132 for 4 hours before harvesting.

(C) Immunopurified SIRT5 proteins were incubated with purified recombinant E1 and ubiquitin (Ub) or mutant Ub-K48 or Ub-K63 in the presence or absence of purified E2 (UBE2D2) and/or TRIM21 proteins as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

(D) Immunopurified SIRT5 proteins were incubated with purified recombinant TRIM21, E1 and E2 (UBE2D2), and ubiquitin (Ub) or mutant Ub-K48R or Ub-K63R proteins as indicated for 45 min at 30℃. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

(E) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting using antibodies specifically against Ub-K48 and Ub-K63. 10 μM MG132 was added for 4 hours before harvesting.

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 4

Figure 4. HAUSP binds to and stabilizes SIRT5, which is repressed in LPS-activated macrophages.

(A) Lysates from 293T cells transfected with HA-HAUSP and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB).

(B, C) Lysates from BMDMs were immunoprecipitated with anti-HAUSP (B), anti-SIRT5 (C) or control IgG antibodies as indicated and then analyzed by western blotting.

(D) IB analysis of WCLs and anti-SIRT5 immunoprecipitates of BMDMs treated with 100 ng/ml LPS for 4 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.

(E) 293T cells were transfected with Flag-tagged HAUSP, mutant HAUSP-CS (C223S) or vector control as indicated for 48 hours. protein expression was determined by western blot analysis using indicated antibodies.

(F) IB analysis of WCLs derived from 293T cells transfected with HA-SIRT5 together with Flag-HAUSP or vector control constructs (left panel). Cells were treated with 100 ng/ml CHX for the indicated time period prior to harvesting. The SIRT5 protein abundance was quantified (right panel).

(G, H) WCLs from BMDMs treated with increasing amounts of P5091(G) or P22077 (H) in the presence or absence of 100 ng/mL LPS were subjected to IB analysis using indicated antibodies (left panel). The SIRT5 protein abundance was quantified (right panel).

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 5

Figure 5. HAUSP deubiquitinates SIRT5, and LPS treatment suppresses this effect in macrophages.

(A) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

(B) IB analysis of WCLs and anti-GST immunoprecipitates of 293T cells transfected with the indicated plasmids in the presence or absence of 100 µM P22077 for 24 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.

(C, D) IB analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

(E) In vitro SIRT5 deubiquitination assay. Ubiquitinated SIRT5 was pulled down from 293T cells cotransfected HA-Ub, and then incubated with purified wild-type HAUSP or the catalytically inactive HAUSP mutant (HAUSP-CS) 30℃ for 90 min. The reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

(F) IB analysis of WCLs and anti-SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 hours. 10 μM MG132 was added for 6 hours before harvesting.

(G) BMDMs transfected with control (-) or HAUSP siRNA for 48 hours were then treated with 100 ng/mL mM LPS for 4 hours, and 10 μM MG132 was added for 4 hours before harvesting. WCLs and anti-SIRT5 immunoprecipitated products were prepared and subjected to IB analysis using indicated antibodies.

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 6

Figure 6. SIRT5 suppresses TRIM21 activity via deacetylation of TRIM21 at Lys351. (A) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids using anti-Flag, anti-Acetyllysine, anti-Succinyllysine, anti-Glutaryllysine and anti-Malonyllysine antibodies

(B) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from 293T cells transfected with control or SIRT5 siRNA using anti-Flag, anti-acetyl Lysine (AcK), anti-succinyl Lysine (SulK), anti-glutaryl Lysine (GluK) and anti-malonyl Lysine (MalK) antibodies.

(C) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

(D) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng/mL LPS or left untreated for 4 hours. 10 μM MG132 was added for 4 hours before harvesting.

(E) Protein expression in SIRT5+/+ and SIRT5-/- BMDMs was determined by western blot analysis.

(F, G) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

(H) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM for 24 hours. 10 μM MG132 was added for 4 hours before harvesting.

(I) Purified TRIM21, TRIM21-K351R and TRIM21-K385R proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and SIRT5 proteins as indicated at 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

(J) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.

Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.