EMBO/soda-nel huggingface dataset:
ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 6

Figure 6. ST3GAL5 promotes TβRI ubiquitination and degradation.

HEK293T cells transfected with Myc-tagged caTβRI, (HA-Ub, and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti-Myc antibody and immunoblot analysis. All groups were treated with MG132 (5 μM) for 6 h.

Ubiquitination of TβRI was detected by IP of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.

Western blot analysis of TβRI expression in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with CHX for the indicated times. Vinculin: loading control.

Quantification of TβRI expression in pLV-EV control- and ST3GAL5-expressing A549-VIM-RFP cells. The results were normalized to the t=0 controls. The data are shown as the mean ± SD of three independent experiments. **, P < 0.01 based on two-way ANOVA.

Working model for UGCG-ST3GAL5-GM3-mediated inhibition of TGF-β signaling via induction of TβRI localization in lipid rafts. The dashed arrow indicates that the SMAD activation-induced decrease in ST3GAL5 expression has not been formally shown.
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 1

Figure 1 - TRF2 overexpression triggers an immunosuppressive tumor microenvironment

A B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice. The percentages of intratumoral CD11bHi GR1Hi MDSCs (n = 10 mice), CD25+ Foxp3+ regulatory T cells (Tregs), or CD25+ Foxp3− activated T cells (n = 5 mice per group) among CD45+ cells were analyzed by fluorescence-activated cell sorting (FACS) and represented as box plots

B-E Schematic representation of the experiment (B). TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, the percentage of immune cells infiltrating the Matrigel plugs of CD11bHi GR1Hi MDSCs (C), the intranuclear staining of pSTAT3 (D), and CD3− NKp46+ and IFN-γ+ among CD3− NKp46+ NK cells (E) were determined by FACS (n = 5 mice per group).

F-I Linear correlation plot of the percentage of IFN- γ + NK cells, the mean of fluorescence intensity (MFI) of pSTAT3 for MDSCs, and the expression of TRF2 (r2= 0,5177; Pearson's correlation, p = 0.003) (F); the mRNA expression level in the matrigel plugs of arg1, pSTAT3 MFI and TRF2 expression (r2= 0,7077; Pearson's correlation, p < 0.0001) (G); the mRNA expression level in the matrigel plugs of tgfβ, pSTAT3 MFI and TRF2 expression (r2= 0,3669; Pearson's correlation, p = 0.0046) (H) and the mRNA expression level in the matrigel plugs of il-10, pSTAT3 MFI and TRF2 expression (r2= 0,7418; Pearson's correlation, p < 0.0001) (I).

Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. In (F-I) Pearson correlation and p values are indicated.
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 2

Figure 2 - TRF2 promotes tumorigenesis and metastasis in an MDSC-dependent manner

A-C B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP). The tumor volume was determined every 2 days using hemi-ellipsoid formula (B). On day 19 of the tumor growth experiment, the percentages of GFP+ cells infiltrating the lungs were determined by FACS and presented as the number of GFP+ cells/mg lung tissue (C).

D-F B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into tail vein of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) every 3 days. On day 21, lung metastasis was analyzed by counting macroscopic metastatic nodules and hematoxylin and eosin (H&E) staining (E and F), scale bars = 500µM.

G-I Tumor-infiltrating immune cells from tumors of the experiment shown in A were analyzed by FACS: MDSCs and CD25+ Foxp3+ Tregs (G), NK cells (H) and CD8+ T cells (I).

J-M BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) as indicated in (J) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS (K-M). Density plot of CD11bHi GR1Hi MDSC staining in control or anti-GR1-treated Matrigel plugs (K). Box plots of the percentages of infiltrating or activated NK cells (CD107a or CD69) (L), of CD11bHi GR1Hi MDSC or NKp46+ NK cells (M) infiltrating the Matrigel plugs assay among the CD45+ cells.

Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. Tumor volume are presented in mm3 along the time in days. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 3

Figure 3 - Cancer cells directly activate MDSCs in a TRF2-dependent manner via the TLR2/MyD88/IL-6/STAT3 pathway that inhibit NK cell degranulation and cytotoxicity.

A-E Analysis of STAT3 phosphorylation by MSC2 cells after 18 hours' co-culture with BJcl2 overexpressing or compromised for TRF2 (A). Histograms of the pSTAT3 mean fluorescence intensity (MFI) (B).

C-E Same experiment than(A-B) +/- inhibitors: anti-MyD88 peptide inhibitor, anti-mouse TLR2 antibody, or lipopolysaccharide (LPS)-RS, an antagonist of TLR4 in (D) and anti-mouse IL-6 or JAK1/2 inhibitor (Ruxolitinib) in (E).

F-H Schematic representation of the MDSC suppression assay (F). TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. After FACS sorting, MSC2 cells were co-cultured for 18h with primed and purified NK cells before a 4h challenge with YAK-1. NK cell degranulation and IFN-γ production were analyzed by FACS (G). Alternatively, a 4h challenge with NIH3T3 cells was performed and the ability of NK cells to kill NIH3T3 cells were determined by colorimetric analysis (AlamarBlue) (H).

Data information: Panels (G and H) show all points with mean ± SEM corresponding to the percentages of each cell population among NK cells (G) or the percentage of specific target lysis (H) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 4

Figure 4 - TRF2 regulates the expression of ITS-associated genes involved in glycocalyx synthesis

A Gene expression analysis by quantitative polymerase chain reaction (qPCR) of TRF2 target genes following TRF2 overexpression or knockdown.

B, C Heatmap of H3K27ac (B) or H3K23me3 (C) peaks on the 4 kilobase (Kb) region around genomic ITSs (ENCODE data for the K562 cell line). The mean intensity of the region is depicted in the histograms. ITSs are clustered depending of the peak mean intensity obtained from H3K27ac or H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) of the 4 Kb region. ITSs detected by ChIP-seq using an anti-TRF2 antibody are shown on the left.

D Specific qPCR for HS3SH4, GPC6, and VCAN ITSs after ChIP using an anti-H3K27ac antibody in TRF2-overexpressing or -knockdown BJcl2 cells. Data represent fold enrichment of the H3K27ac level compared with the total H3 level.

Data information: data represent n = 3 independent experiments with mean ± SEM
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 5

Figure 5 - HS3ST4, GPC6, and VCAN are effectors of the MDSC dependent oncogenic properties of TRF2

A Schematic representation of the Matrigel plug assay with BJcl2 cells knockdown for TRF2 target genes. Immune cells infiltration within the Matrigel plugs was determined by FACS.

B-E Percentage of CD11bHi GR1Hi MDSCs (B), CD3- NKp46+ NK cells (C), NKp46+ CD107a+ NK cells (D) or CD69+ NK cells (E)

F-H Analysis of STAT3 phosphorylation by MSC2 cells after 18 hours co-culture with BJcl2 overexpressing or compromised for TRF2 and the target genes (F). Histograms of the pSTAT3 mean fluorescence intensity (MFI) of MSC2 cells when TRF2 was overexpressed and the target gene knocked down (G) and when both TRF2 and its target gene were knocked down (H).

I Histograms of tumor weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 cells overexpressing or knocked down for HS3ST4 or GPC6

Data information: In (B-E), data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ or NK cells (n = 8 mice per group; *p < 0.05 and ***p < 0.001; Mann-Whitney test). In (G and H), MFI of n=3 independent experiment is normalized to controls and represented as fold change of MFI (n = 3 independent experiments; *p < 0.05 and **p < 0.01; Student's t-test). In (I), data are represented as mean +/- SEM (n = 8 mice per group; *p < 0.05, and **p < 0.01; Mann-Whitney test).
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 6

Figure 6 - TRF2 changes glycocalyx structure, which triggers MDSC activation

A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody.

B Atomic Force Microscopic (AFM) analysis of glycocalyx and cell stiffness and glycocalyx length.

C AFM images showing the Modulus heatmap (stiffness) in GFP-expressing or TRF2-overexpressing cancer cells. At each of the 128x128 force curve points, corresponding to an area of 10 μm2, the AFM software automatically displays the Sneddon modulus analysis while scanning the sample. The color bar on the left of the images shows a qualitative representation of the elastic map.

D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3).

Data information: In (A), histograms representative (left panels) and MFIs from three independent experiments (*p < 0.05 and **p < 0.01; Student's t-test) are shown +/- SEM. In (B), Box plots Min to Max showing all points with median represent four independent experiments, each performed using at least 20 cells (*p< 0.05; Mann-Whitney test). For more details on the analysis procedure see Method section. In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 7

Figure 7 - TRF2 upregulation enhances the response to chemotherapies targeting MDSC

A Kaplan-Meier curves for overall survival of chemotherapy-treated patients according to the TRF2 expression level (KM ploter, http://kmplot.com/analysis/index.php?p=background).

B-E Schematic representation of the mouse experiment. Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. The tumor volume was determined every 2 days using a caliper, and growth curves over time (C) and tumor volume were determined on day 19 and represented using Box plots Min to Max showing all points with median (D). Overall survival was also determined (E).

Data information: in (A), the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated. In (B-C), data represent mean values +/- SEM (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test). In (D), data are represented by Box plots Min to Max showing all points with median (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test)
Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2 (2019)
Figure 8

Figure 8 - TRF2 upregulation in human malignancies is associated with a poor prognosis, high expression of HSPG genes and MDSC infiltration.

A Kaplan-Meier curves for overall survival of patients at all stages of cancers (breast, gastric, ovarian, and lung cancer) depending on TRF2 expression level. (KMplot, http://kmplot.com/analysis/index.php?p=background).

B-D The overall survival of gastric cancer patients (all stages) was analyzed depending on TRF2 and its targets genes expression level (HS3ST4, GPC6 and VCAN collectively named the HSPG genes) or with MDSC level (Lo or Hi), determine as the mean expression level of CD33 and C5AR, using KMplot, http://kmplot.com/analysis/index.php?p=background.

E Repartition of number of gastric cancer patients depending on TRF2 and MDSC classification.

F Overall survival of gastric cancer patients (all stages) analyzed depending on TRF2, HSPG genes (HS3ST4, GPC6 and VCAN) and MDSC level (Lo or Hi).

Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.

Tables

Table 1. List of the 483 ITS identified including their localization and associated genes

Table 2. Pathway analysis (Reactome) from the 483 ITS

Table 3. ITS number and associated epigenetic mark during mammal's evolution

Table 4. ChIP-seq analysis for H3K27ac mark

Table 5. List of the TRF2 mutations found in cancer patients (TCGA analysis)

Table 6. Median of survival depending on TRF2 expression (KMplot analysis)

Expended View Figure Legends:
Zika virus inhibits type-I interferon production and downstream signaling (2016)
Figure 1

Figure 1. ZIKV inhibits induction of the IFN response

A549 cells were transfected with 2 μg of poly(I:C) for 6 h and then infected with ZIKV (MOI = 3) for 18 h (pre‐treatment) or infected with ZIKV first for 6 h and then transfected with poly(I:C) for 12 h (post‐treatment). Levels of ZIKV genomic RNA were determined by qRT–PCR. Values are expressed as mean ± standard error. ***P < 0.001 (Student's t‐test), N = 3.

A549 cells infected with ZIKV (MOI = 3) for 6 h were transfected with 2 μg poly(I:C) for 12 h after which levels of Ifnb and Ifit1 mRNA were determined by qRT–PCR. Values are expressed as mean ± standard error. ***P < 0.001 (Student's t‐test), N = 3.

A549 cells were infected with ZIKV (MOI = 5) for 16 h and then transfected with an IFIT1 promoter‐driven firefly luciferase plasmid (pGL3B/561) and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N = 2.

HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later, they were transfected with an IFIT1 promoter‐driven firefly luciferase plasmid (pGL3B/561) and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test), N = 4.

A549 cells were infected with ZIKV (MOI = 5) for 16 h and then transfected with the indicated promoter‐driven firefly luciferase plasmids and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N = 2.

HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter‐driven firefly luciferase plasmids and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 0.4 μg of poly(I:C). Twenty‐four hours later, cell lysates were harvested and subjected to luciferase assay. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.
Zika virus inhibits type-I interferon production and downstream signaling (2016)
Figure 2

Figure 2. ZIKV infection blocks IFN signaling.

A. A549 cells were treated with IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN–γ (10 U/mL) for 6 hrs after which they were infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment). Alternatively, cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs (Post-treatment). Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student’s t-test); N=3

B. A549 cells were infected with ZIKV (MOI=3) for 6 hrs prior to addition of IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN–γ (10 U/mL) for 12 hrs. Total RNA was isolated and levels of ifit1 RNAs were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student’s t-test); N=3

C. A549 cells were infected with ZIKV (MOI=5) for 6 hrs and then transfected with ISRE- or GAS- promoter-driven firefly luciferase together with a constitutively active renilla luciferase construct. Sixteen hours later, cells were treated with IFN-α (100 U/ml) or IFN-γ (10 U/ml) for 2 hrs. Cells lysates were harvested and relative luciferase activity from ISRE- and GAS-promoters were determined. Values are expressed as mean ± standard error. *P<0.05 (Student’s t-test); N=3

D. HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK). After 24 hrs they were treated with IFN-α (100 U/mL) for 10 hrs. Cell lysates were then harvested and subjected to luciferase assays. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05 (Student’s t-test); N=3
Zika virus inhibits type-I interferon production and downstream signaling (2016)
Figure 3

Figure 3. ZIKV infection causes degradation of human STAT2.

A. A549 cells were infected with ZIKV (MOI=5) for 24-48 hrs after which cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

B. A549 cells were infected with ZIKV (MOI=5) for 24 hrs and then treated with DMSO, MG132 (20 µM) or epoxomicin (Epox) (400 nM) for 12 hrs. Cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

C. A549 cells (MOI=2) or Human primary fetal astrocytes (HFA) (MOI=5) were infected with ZIKV for 24 hrs and 48 hrs respectively, after which IFN-α (100 U/mL) was added for 2 hrs. Cells were then fixed and processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. Dashed line white circles indicated ZIKV-infected cells. Representative panels are shown. The experiment was repeated two times.

D. Mouse embryonic fibroblasts (MEF) were infected with ZIKV (MOI=5) for 48 hrs after which IFN-α (200 U/mL) was added for 2 hrs. Samples were processed for immunofluorescence assay as described in panel C. Representative panels are shown. The experiment was repeated two times.

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times. The ZIKV replication was measured by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student’s t-test); N=3
Zika virus inhibits type-I interferon production and downstream signaling (2016)
Figure 4

Figure 4. NS5 protein interacts with human STAT2 and induces its degradation.

A. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG-tagged WT or mutant NS5 proteins. At 48 hrs post-transfection cells were treated with IFN-α (100 U/mL) for 2 hrs and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. NS5 positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.

B. Quantification of nuclear STAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D.= not detected. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student’s t-test); N=3

C. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKV NS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.

D. A549 cells were transfected with plasmids encoding FLAG-tagged WT, mutant NS5 proteins or FLAG alone for 48 hrs. Cells were harvested and process for immunoprecipitation (IP) using rabbit anti-STAT2 followed by SDS-PAGE and immunoblotting. Arrows indicate co-immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 2

Fig. 2. Spermine metabolized to acrolein promotes lysosomal distribution towards MTOC and facilitates autophagy.

a, b. SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) and 40 μM acrolein for 2 h (b). Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Scale bar, 10 µm. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right). *** p<0.0001; N.S., not statistically different (Dunnett's test, vs NT) Four images were quantified per each condition. Values are mean ± SD. At least three experiments were replicated.

c. SH-SY5Y cells pre-treated with 1 mM aminoguanidine (AG) for 1 h were treated with spm for 4 h. Cells were fixed and stained with an anti-LAMP2 antibody. Scale bar, 20 µm.

d. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with an anti-γ-tubulin (green) antibody to detect MTOC and an anti-LAMP2 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.

f. SH-SY5Y cells stably expressing GFP-LC3 were pre-treated with 10 μg/ml E64D plus pepstatin A for 2 h and then treated with 40 μM acrolein for an additional 2 h. Cells were fixed and stained with an anti- LAMP2 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

g. SH-SY5Y cells stably expressing GFP-LC3 were pre-treated with 10 μg/ml E64D plus pepstatin A for 2 h and then treated with 50 μM spm for an additional 4 h, followed by CLEM analysis. Structures containing GFP were identified by confocal microscopy in fixed cells and then relocated after Epon embedding and imaged by electron microscopy. Lysosome-like structures and centrosomes are indicated by white and red arrows, respectively. Boxed regions (1, 2) are enlarged on the right.

h. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.