EMBO/soda-nel huggingface dataset:
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 3

Figure 3. Deficiency of Ang in myeloid cells accounts for the aggravated colitis.

(A, B) (A) Body weight loss and (B) DAI of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.

(C-F) (C) Colon length, and (D) serum FITC-Dextran level, (E) representative HE staining image, and (F) histopathological score of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.

(G) Ang mRNA expression in mouse lymphoid and myeloid cells; data represent two independent experiments (n = 2).

(H, I) (H) Body weight loss and (I) DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.

(J-M) (J) Colon length, (K) serum FITC-Dextran level, (L) representative HE staining image, and (M) histopathological score of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.

Data information: Scale bar, 50 μm; data are shown as mean ± SEM; Kruskal-Wallis with Dunn's multiple comparisons test (A, B and F), ANOVA with Dunnett's multiple comparisons test (C and D) and two-tailed unpaired student's t test (H, I, J, K and M) are used to determine statistical significance (*P<0.05, **P<0.01, ***P<0.001).
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 4

Figure 4. ANG-PLXNB2 axis is essential for mitigating DSS-induced colitis in mice.

(A, B) (A) Quantitative mRNA expression and (B) western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9).

(C) Representative images showing immunofluorescence staining of ANG, PLXNB2 and macrophage surface marker F4/80 in frozen colonic section from WT mice.

(D) Representative images showing immunofluorescence staining of GFP and PLXNB2 in frozen colonic section from WT mice infected with adeno-associated virus (AAV9).

(E) PLXNB2 expression in IECs from the AAV9-infected WT mice (n = 3).

(F, G) (F) Body weight loss and (G) DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.

(H-K) (H) Colon length, (I) serum FITC-Dextran level, (J) representative HE staining image, and (K) histopathological score of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.

Scale bar, 50 μm; data are shown as the mean ± SEM; Mann Whitney test (A, G and I) and two-tailed unpaired student's t test (E, F, H and K) are used to determine statistical significance (*P<0.05, **P<0.01; ***P<0.001).
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 5

Figure 5. ANG protects intestinal epithelial integrity by enhancing IEC survival and proliferation.

(A, B) (A) Representative images showing cleaved-caspase 3 staining and (B) the number of apoptotic cells in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point; arrows indicate cleaved-caspase 3 positive cells; two fields per colon section are quantified.

(C and D) Cell apoptotic status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.

(E, F) (E) Representative images showing Ki67 staining and (F) the number of proliferating cells per crypt in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point; five representative crypts per colon section are quantified.

(G and H) Cell-cycle status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.

(I, J) (I) Representative images showing cleaved-caspase 3 staining and (J) the number of apoptotic cells in colonic section from Angfl/fl and LyzMCre;Angfl/fl, or AAV-shControl- and AAV-shPlxnb2- treated colitis mice on day 4 (n = 3); arrows indicate cleaved-caspase 3 positive cells; two fields per colon section are quantified.

(K, L) (K) Ki67 staining and (L) the number of proliferating cells per crypt in representative colonic section from Angfl/fl and LyzMCre;Angfl/fl, or AAV-shControl- and AAV-shPlxnb2- treated colitis mice on day 4 (n = 3); five representative crypts per colon section are quantified.

Data information: Scale bars: 50 μm; data are shown as mean ± SEM; Mann Whitney test (B and F) and two-tailed unpaired student's t test (D, H, J and L) are used to determine statistical significance (*P<0.05, **P<0.01, ***P<0.001).
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 6

Figure 6. ANG enhances IEC survival and proliferation via mediating different RNA production.

(A) PLXNB2 expressions in HCT116 (including wild type HCT116 (HCT116WT) and ANG knockout HCT116 (HCT116KO)) and THP-1 as detected by flow cytometry analysis. The upper three dash lines represent the intensity of unstained negative control of these cells, while the solid lines represent the intensity of PLXNB2 antibody staining.

(B) Secreted ANG level in conditioned medium from THP1, HCT116WT or HCT116KO (n = 3).

(C) The interaction between ANGWT, ANGR66A or ANGK40I with PLXNB2 in HCT116KO detected by co-immunoprecipitation assay.

(D) THP-1-derived ANG translocating into HCT116KO through PLXNB2 in co-cultured system as showed by western blotting.

(E) Flow cytometry analysis of HCT116KO stained with Annexin V and PI following different treatments as indicated.

(F) tiRNA and 5'-tiRNAAla production in HCT116KO following different treatments as indicated.

(G) The effect of 5'-tiRNA knockdown or overexpression on TNF-α-induced apoptosis in ANGWT- or ANGK40I-treated HCT116KO.

(H) The interaction between 5'-tiRNA and cytochrome c (Cyt c) in HCT116 cells as detected by RIP.

(I) Caspase activity of HCT116KO following different treatments as indicated.

(J) Cell viability of HCT116KO following different treatments as indicated.

(K) Detection of 47S pre-rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated.

(L) tiRNA and 5'-tiRNAAla production in IECs isolated from WT and Ang-/- colitis mice at indicated time point.

(M) 47S pre-rRNA transcription level in IECs from WT and Ang-/- colitis mice at indicated time point.

Data information: Neamine, a blocker of ANG and PLXNB2 interaction; siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs; si5'-tiRNA: tiRNA-targeting siRNA; ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation; data are shown as mean ± SEM, and represent at least 3 independent experiments in E, G, H, I and J. ANOVA with Dunnett's multiple comparisons test (B, I and J) and Mann Whitney test (H) are used to determine statistical significance (**P<0.01, ***P<0.001).
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 7

Figure 7. Recombinant ANG protects mice from DSS-induced colitis.

(A) Schematic diagram of the ANG prevention model. Mice (n = 8) were pre-treated with 1.25 mg/kg recombinant ANG protein and then subjected to DSS induction.

(B) Western blotting of ANG in colonic tissue from WT mice (n = 2) at indicated time point in the ANG prevention model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.

(C, D) (C) Body weight loss and (D) DAI of the pre-treated WT colitis mice (n = 8).

(E, H) (E) Colon length, (F) serum FITC-dextran level, (G) representative HE staining image, and (H) histopathological score of colon section from the pre-treated WT colitis mice on day 8 (n = 8).

(I) Schematic diagram of the ANG treatment model. Mice (n = 8) were treated with DSS for 7 days, and simultaneously administrated with 1.25 mg/kg recombinant ANG during day 4-6.

(J) Western blotting of ANG in colonic tissue from WT (n = 2) mice at indicated time point in the ANG treatment model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.

(K, L) (K) Body weight loss and (L) DAI of the treated WT colitis mice (n = 8).

(M-P) (M) Colon length, (N) serum FITC-dextran level, (O) representative HE staining image, and (P) histopathological score of colon sections from the treated WT colitis mice on day 8 (n = 8).

Data information: Scale bar, 50 μm; data are shown as mean ± SEM; two-tailed unpaired student's t test (C, E, F, K, M, N and P) and Mann Whitney test (D, H and L) are used to determine statistical significance (* P<0.05).

Expanded view figure legends
Opposing activities of IFITM proteins in SARS-CoV-2 infection (2020)
Figure 1

Figure 1: Human IFITMs inhibit SARS-CoV-2 Spike-pseudotyped lentivirus infections.

(A, B) HEK293T cells transduced with the indicated FLAG-tagged IFITM expression constructs or vector control plus transient transfection of ACE2 were infected with HIV-1expressing a luciferase reporter gene pseudotyped with (A) SARS-CoV-2 Spike (HIV-SARS-2) or (B) SARS-CoV Spike (HIV-SARS-1). Measurement of luciferase activity 72 h post infection served as a readout for infection. Luciferase readings were normalized to vector control, which was set to a value of 100. Bars in (A and B) represent averages with individual data points from 3-5 independent experiments shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test. Not indicated in (A), IFITM1 was also significantly different from IFITM2 and IFITM3.

C) Representative example flow cytometry plots from cells transfected as in A/B demonstrate high transfection efficiency and expression of each of the FLAG-tagged IFITMs.

D) Caco-2 cells were transfected as in (A/B) and infected with HIV-SARS-2. Luciferase readings at 72 h post infection were normalized to vector control, which was set to a value of 100. Bars represent averages with individual data points from 4 independent experiments shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.

E) Caco-2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h. Western blotting of cell lysates is shown.

F) Cells as in (E) were infected with HIV-SARS-2. Luciferase readings at 48 h post infection were normalized to vector control, which was set to a value of 100. Bars represent averages with individual data points from 3-7 independent experiments shown as circles. Error bars represent SD. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test for the comparisons indicated by lines.
Opposing activities of IFITM proteins in SARS-CoV-2 infection (2020)
Figure 6

Figure 6: Mutation of the human IFITM3 endocytosis motif converts IFITM3 into an enhancer of SARS-CoV-2 Spike-mediated cell-to-cell membrane fusion.

U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye.

A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm.

B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.
Opposing activities of IFITM proteins in SARS-CoV-2 infection (2020)
Figure 7

Figure 7: IFITM3 restriction of SARS-CoV-2 infection requires the IFITM3 amphipathic helix while enhancement of infection does not.

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h.

A) Transfected HEK293T-ACE2-GFP cells were infected with SARS-CoV-2 (MOI 1) for 24h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each transfection condition.

B) Graph depicts normalized infection percentage measurements from 3 independent experiments, each performed in triplicate, from cells infected as in (B). Bars represent averages with individual data points shown as circles. Error bars represent SD. The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test. NS, not significant. Not indicated, additional comparisons of interest with p<0.05 include IFITM3 (-) vs. IFITM3-Y20 (-), IFITM3∆59-68 (-), and IFITM3-S61A,N64A,T65A (-).

C) Flow cytometry histograms showing expression of each of the IFITM constructs with the dotted line indicating gating that was used for examining percent infections in IFITM-expressing cells.
SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break pathway choice. (2018)
Figure 1

Figure 1. Identification of novel REV7 interactors relevant for the NHEJ pathway.

(A) Venn Diagram representing the distribution of proteins identified by both the BioID and the standard AP/MS of REV7, with or without DNA damage (NCS).

(B) Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits.

(C) U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data is presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05, **p<0.005, ***p<0.0005
SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break pathway choice. (2018)
Figure 2

Figure 2. SHLD2 plays a critical in DNA repair.

(A) Pathway enrichment analysis on genes co-evolving with SHLD2 in Mammalians and Vertebrates using a phylogenetic profiling approach followed by an Enrichr-based analysis.

(B) Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment. Data are represented as a box and whiskers graph where the box tends from the 25th to the 75th percentiles while the whiskers are drawn down to the 10th percentile and up to the 90th. Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05, **p<0.005, ***p<0.0005. For each condition, at least 50 cells were analyzed.

(C) U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Data are represented as a bar graph showing the mean +/- SD. Significance was determined by two-way ANOVA followed by a Bonferroni test. *p<0.05, **p<0.005.

(D) Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05.

(E) Similar to (D), except that cells were exposed to increasing doses of UV radiation. Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05.
SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break pathway choice. (2018)
Figure 3

Figure 3. SHLD2 is recruited and accumulates at DNA damage sites.

(A) Schematic representation of SHLD2 and the different mutants used in this study. Each putative structural domain of SHLD2 is represented.

(B) U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.

(C) U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm.

(D) Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.

(E) Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus. Data are represented as a box-and-whisker plot in the style of Tukey. At least 100 cells per condition were counted. Significance was determined by non-parametric test followed by a Kruskal-Wallis test. *p<0.005, **p<0.0005.

(F) Schematic representation of the site-directed generation of DSB by the restriction enzyme AsiSI (Top). 293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom). Data is presented as the mean ± SEM (n=3). Significance was determined by two-way ANOVA followed by a Sidak test. *p<0.05.

(G) U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.
SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break pathway choice. (2018)
Figure 5

Figure 5. SHLD1 cooperates with SHLD2 and REV7 to promote NHEJ and restrict HR.

(A) Schematic Representation of CRISPR/Cas9-based Genome-Wide Screen under Doxorubicin treatment.
(B) Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.

(C) 293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.

(D) U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data is presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.005, **p<0.0005.

(E) CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis. Data are presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05, **p<0.005.

(F) U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data are presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.005, **p<0.0005.
Novel tau biomarkers phosphorylated at T181, T217 or T231 rise with subtle changes in Aβ pathology (2020)
Figure 1

Figure 1. P-tau biomarkers changes with age

A-E Scatter plots showing the association of each of the p-tau biomarkers with age in the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n = 131) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for sex. Additionally, we computed the 'Age x Aβ status' interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.
Novel tau biomarkers phosphorylated at T181, T217 or T231 rise with subtle changes in Aβ pathology (2020)
Figure 2

Figure 2. Effect of Aβ pathology (CSF Aβ42/40) on p-tau biomarkers

(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n =131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). P-values were assessed by a one-way ANCOVA adjusted for age and sex.

(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of CSF Aβ42/40. The horizontal-axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P-values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cutoff. Participants were also colour-coded based on the Aβ PET CL scale (≤12CL, black; >12CL, turquoise; Aβ PET non-available, grey).

Data information: Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; n.a. non-available; p-tau, phosphorylated tau.