EMBO/soda-nel huggingface dataset:
Convergence of vitamin D and retinoic acid signalling at a common hormone response element (2005)
Figure 1

Identification of ER8 elements in 1,25‐dihydroxyvitamin D3 and RA target genes. (A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor. (B) Reverse transcription–PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.
Convergence of vitamin D and retinoic acid signalling at a common hormone response element (2005)
Figure 2

Characterization of in vitro and in vivo p19ink4d ER8 element function. (A) Electrophoretic mobility shift assay (EMSA) of binding of vitamin D receptors (VDRs)/retinoid X receptors (RXRs) to the p19ink4d ER8, with binding to the mouse osteopontin (mop) DR3 vitamin D responsive element as a control, along with a comparison of binding to ER6 and ER8 motifs (right‐hand panel). (B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control. (C) Chromatin immunoprecipitation (ChIP) analysis of binding of VDRs and RARs to the p19ink4d ER8 (region 1) and RNA polymerase II (POLII) binding to the transcription start site. (D) ReChIP analysis confirms the presence of VDR/RXRs and RAR/RXRs but not of VDR/RARs on the p19ink4d ER8. (E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 1,25‐Dihydroxyvitamin D3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.
Convergence of vitamin D and retinoic acid signalling at a common hormone response element (2005)
Figure 3

1,25‐dihydroxyvitamin D3 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription–PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 1,25‐dihydroxyvitamin D3 (1,25D3) or retinoic acid (RA), as indicated. (C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase (CDK)4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below. (D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.
Convergence of vitamin D and retinoic acid signalling at a common hormone response element (2005)
Figure 4

Knockdown of p19ink4d and p27kip1 in SCC25 cells. (A) Short interfering RNAs (siRNA) knockdown of p19ink4d messenger RNA and protein in SCC25 cells. SiRNAs were directed against p19ink4d, scrambled p19ink4d (Scr.) or cyclophilin (cyc.) controls. (B) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 (top) and p19INK4D (bottom). (C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated. (E) Loss of p19INK4D enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19INK4D siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3). (F) Loss of p19INK4D induces lysosomal fusion. Analysis of lysosomal fusion (autophagosomes) in SCC25 cells treated as in (E), stained for incorporation of lysotracker red. Nuclei were counterstained with Hoechst (blue).
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 1

Figure 1. EGFL7 expression in glioma

(A) The egfl7 gene (about 13.8 kb) comprises 10 exons with 7 of them encoding for the EGFL7 cDNA (lower left). Intron 7 harbors the parasitic miRNAs (miR) 126 and 126* (lower right). (B) Methylation arrays of primary glioblastoma (GBM) specimens revealed that CpG island 1 (egfl7 promoter) was mostly unmethylated with the exception of some samples in the RTKII subgroup (*). CpG island 2 (miR-126 promoter) was found methylated in most cases except the G34R subgroup (**). G34R and K27M - GBMs with mutations in a gene for the H3.3 histone variant; RTKI and II - GBMs with a mutation in the receptor tyrosine kinase type I (proneural subtype) and II (classical subtype); MES - GBMs with a deleterious mutation in the NF1 gene (mesenchymal subtype); IDH1 - GBMs with a mutation in isocitrate dehydrogenase 1. (C) Quantification of human EGFL7 by qRT-PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM-derived organotypic spheroids (n = 3; Mann-Whitney U-test; mean ± SEM); B - GBM biopsy; G - generation. (D) Alignment of EGFL7 expression along with stromal (VWF, VEGFR1, VEGFR2, ACTA2, CD248, ITGAX, AIF1, CD68, CD4, and PTPRC) or tumor markers (EGFR, PDGFRA, VEGFA, VIM, and NES) in primary GBM biopsies or GBM-derived organotypic spheroids (PDX xenografts). (E) Immunohisto­chemical (IHC) analyses revealed EGFL7 expression in blood vessels (arrow) and neurons (arrowheads) of healthy gray matter. Expression in malignant brain tumor specimens was restricted to blood vessels, in particular with a large diameter (arrows). Scale bar represents 50 µm. (F) Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25-40% EGFL7-positive blood vessels (n = 13 astrocytoma WHO° grade II, n = 25 astrocytoma WHO° grade III, n = 24 GBMs WHO° grade IV).
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 2

Figure 2. EGFL7 affected tumor growth and reduced overall survival

(A) Schematic presentation of intracranial tumor implantation. (B) Overexpression of human EGFL7 (hE7) or murine EGFL7 (mE7) increased tumor growth (n = 9; one-way ANOVA). (C) Immunocompetent mice bearing GL261 tumors. Representative MRI images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 34 d (control) vs. 29.5 d (hE7) vs. 31.5 d (mE7); n = 8; log-rank test). (D) Immunodeficient mice bearing U87 tumors. Representative magnetic resonance images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 70 d (control) vs. 42.5 d (hE7) vs. 40 d (mE7); n = 8; log-rank test). (E) miR-126 KO did not affect overall survival of GL261 tumor-bearing mice (n = 8). (F) EGFL7 KO prolonged overall survival of GL261 tumor-bearing mice as compared to wild-type (WT) littermates (44 d in KO vs. 35.5 d in WT; n = 8 each; log-rank test). (G) Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus-based approach. Knock-down of EGFL7 (shE7_1 or shE7_2) prolonged the median survival time of glioma-bearing mice (68 d or 69 d) as compared to scrambled control (shScr; 56 d; n = 6; log-rank test). (H) The blood vessel-specific and miR126-independent KO of EGFL7 in EGFL7fl/fl;Cdh5-CreERT2 mice prolonged the overall survival of GL261 tumor-bearing mice as compared to WT littermates (38 d in KO vs. 33 d in WT; n = 7 for KO; n = 5 for WT; log-rank test). All data are presented as mean ± SEM. Scale bars represent 5 mm.
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 3

Figure 3. EGFL7 promoted density and maturation state of glioma vessels

(A+B) CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 (hE7)- or murine EGFL7 (mE7)-positive tumors (n = 3; one-way ANOVA). (C+D) T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm. Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of (E+F) PDGFRβ (pericytes), (G+H) SMA (smooth muscle cells), or (I+J) Col IV (basement membrane) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 4

Figure 4. EGFL7 affected angiogenic sprouting in dependence of integrin α5β1

(A) Structural analysis of recombinant EGFL7 by transmission electron microscopy (TEM) showed aggregates with a fibrillar structure of varying sizes. Black scale bar left represents 50 nm. Higher magnifications depict rod-like EGFL7 particles. Black scale bar right represents 15 nm. White scale bar represents 5 nm. (B+C) Positive charged amino acid residues at the C and negative charges at the N terminus make EGFL7 a polar molecule with a preference towards oligomerization. (D) Angiogenic sprouting of primary human umbilical vein endothelial cell (HUVEC) spheroids embedded in a collagen-based matrix was induced by the application of EGFL7 (E7), fibronectin (Fn), or a combination of both (upper row). Co-application of an integrin α5β1 blocking antibody (anti-α5β1) reduced sprouting in all cases but control (lower row). Scale bar represents 100 µm. (E) E7, Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid, an effect blocked by anti-α5β1 (n = 3; one-way ANOVA (inter-group differences) and Mann-Whitney U-test (intra-group differences); mean ± SEM). (F) Measurement of Rho GTPase signaling downstream of integrins. Quantification of (G) Cdc42, (H) Rac1 and (I) RhoA activation by immunoblotting subsequent to seeding HUVECs on E7, Fn or a combination of both. Preferentially, E7 activated Cdc42 and Fn activated Rac1, but both proteins antagonized each other. None of the treatments affected RhoA signaling (n = 3; one-way ANOVA). IP-immunoprecipitation, AU-arbitrary units.
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 5

Figure 5. EGFL7 increased surface expression of integrins α5β1 and αVβ3

(A) Schematic of the experimental setup to test for surface expression of integrins α5β1 and αVβ3 in human umbilical vein endothelial cells (HUVEC). All surface molecules were marked by biotiny­lation followed by immunoprecipitation (IP) and western blot specifically detecting each integrin α or β subunit. Cells have not been permeabilized in either case to avoid staining of intracellular proteins. Alternatively, antibodies specific for both integrins were applied and detected by FACS. (B-D) Surface biotinylation assays verified increased surface expression of αV and β3 upon appli­cation of E7 but not Fn (n = 3; one-way ANOVA). (E-G) Surface expression of α5 and β1 was found increased upon treatment with E7 or Fn and was further enhanced by combination of both (n = 3; one-way ANOVA). (H) Ectopic expression of murine EGFL7 (mE7) in GL261 mouse glioma cells followed by intracranial implantation into the striatum of immune-competent mice reduced overall survival but was rescued upon treatment with an integrin α5β1-inhibiting antibody (n = 6; log-rank test). Data presented as mean ± SEM. TCL-total cell lysate, AU-arbitrary units.
EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors (2018)
Figure 6

Figure 6. Anti-EGFL7 treatment for the treatment of glioma in vivo

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. (A+B) CD31 staining revealed that blocking of EGFL7, VEGF, or a combination of both lead to decreased tumor vessel density (n = 3; one-way ANOVA). Staining for (C+D) PDGFRβ (n = 3; one-way ANOVA), (E+F) SMA (n = 3; one-way ANOVA), or (G+H) Col IV (n = 3; one-way ANOVA) revealed decreased amounts of blood vessel-associated pericytes, smooth muscle cells and Col IV in the basement membrane of blood vessels upon treatment with anti-EGFL7, anti-VEGF and most significantly, a combination of both antibodies. (I) This resulted in an increased intratumoral vessel permeability as measured by T1-weightened MRI analyses of extravasating Gadovist (n = 6; one-way ANOVA). (J) Treatment with anti-EGFL7 (47 d), anti-VEGF (51 d), or a combination of both antibodies increased the median survival time of glioma-bearing Rag1-/- mice (58 d) as compared to isotype-treated controls (37 d; n = 6; log-rank test). (K) Treatment of glioma-bearing NOD SCID mice with temozolomide (TMD) as a chemotherapeutic agent and a combination of anti-EGFL7 and anti-VEGF antibody increased the median survival time (Combo; 87 d; n = 5; log-rank test) as compared to anti-VEGF alone (80 d; n = 5; log-rank test) or isotype-treated controls (69 d; n = 5; log-rank test). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 1

Figure 1. Functional mapping of DDX60 antiviral domains and interrogation of anti-HCV activity.

(A) Schematic of DDX60 protein with putative functional domains. Helicase ATP binding type I domain (amino acids 785 - 921) and C-terminal helicase domain (amino acids 1291-1331) are shown as larger boxes in linear DDX60 schematic. Amino acids are numbered below. Putative functional motifs (I, II, III, and VI) and mutations made are annotated. The amino acids in bold as well as N and C-terminal regions were interrogated in antiviral assays.

(B) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.

(C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES.

(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B.

Data information: For (C) and (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 2

Figure 2. In cell reporter assay for reporters translationally driven by different internal ribosome entry sites.

(A) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5' cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5' cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5' cap-driven Rluc units given either: equal translation of 5' cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5' cap-driven Rluc (bottom).

(B) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5' cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Raw data is shown in Fig EV2.

Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 3

Figure 3. Viral replication assays for a panel of IRES containing viruses.

(A-E) (A, B, E) Multi-cycle infection assays with type I and type II IRES containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) encephalomyocarditis virus, (B) poliovirus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV-IRES-PV) at MOI 0.001. Supernatants were collected 24 hours post infection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV-IRES-PV) and titers determined via plaque assay on HeLa cells. (C, D) Single-cycle infection assays with type I and type II IRES containing viruses. BHK-J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) foot and mouth disease virus, or (D) bovine enterovirus-1 at MOI 1. Supernatants were collected 5 hours post infection (hpi) and titers determined via plaque assay on BHK-21 clone 13 cells.

Data information: Data shows mean ± SEM percent infectious titers relative to Fluc from at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against DDX60 E890A. PFU data is shown in Fig EV3.
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 4

Figure 4. mRNA abundance for reporters translationally driven by different internal ribosome entry sites, and interferon-stimulated response element activity in the presence of DDX60.

(A, B) HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5' cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 16 hours later. (A) Abundance of luciferase reporter mRNAs assayed using RT-qPCR. Data is representative of at least 3 biological replicates from experiments performed on separate days. ns, not significant using one sample t-test comparing mean fold changes to theoretical mean of 1. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. (C, D) HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (C) or western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (D). Quantification of DDX60 and RIG-I band intensities relative to ß-actin are shown below each respective lane in (D).

Data information: (A, B) Mean ± SD from at least n=3 biological replicates; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells. ns, non-significant. (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60 wt or RIG-I + DDX60 E890A transfected cells. (D) representative western blot from (C).
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 5

Figure 5. Polysome profile of type II IRES Fluc mRNA in the presence of DDX60.

(A) Schematic of polysome profiling strategy. HEK293T cells were transfected with DDX60 wt or DDX60 E890A (negative control). 48 hours post transfection, cells were transfected with in vitro transcribed 5' cap- or EMCV IRES-driven Fluc mRNA constructs. 16 hours post transfection, duplicate samples were treated with 200 μM puromycin for 20 minutes as positive controls for decrease in polysomes. Cells were treated with 100 μg/mL of cycloheximide for 15 minutes to arrest polysomes and subjected to polysome profiling by ultracentrifugation through 15%-50% sucrose gradients. Amount of Fluc reporter mRNA from polysome fractions was determined by RT-qPCR.

(B) Effect of DDX60 on 5' cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes.

Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n=3 biological replicates. Full polysome profiles are shown in Fig EV5.