EMBO/soda-nel huggingface dataset:
uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils (2021)
Figure 2

Figure 2. Mechanisms underlying uPA-PAI-1-dependent neutrophil trafficking.

(a) Deposition of uPA or PAI-1 (green) in the cremaster muscle of WT mice as assessed separately ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of TNF or saline, PECAM-1/CD31+ postcapillary venules (red) and Ly-6G+ neutrophils (purple) are depicted. Representative images are shown (scale bar: 50 µm).

(b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).

(C, D) (c) Production of TNF was analyzed in mouse RAW 264.7 macrophages and (d) surface expression of ICAM-1/CD54, VCAM-1/CD106, uPA, and PAI-1 on mouse bEnd.3 microvascular endothelial cells as assessed in vitro by multi-channel flow cytometry upon exposure to recombinant murine uPA-PAI-1, TNF, or saline, quantitative data are shown (mean±SEM for n=4 experiments per group; #p<0.05 vs. saline; One-way ANOVA / t test).

(E, F) (e) Binding of ICAM-1/CD54-Fc or VCAM-1/CD106-Fc to primary mouse neutrophils and (f) binding of conformation-specific mABs to primary human neutrophils ('mAB 24' recognizing high-affinity conformation, 'Kim127' recognizing intermediate and high affinity conformation of β2 integrins) as assessed upon exposure to recombinant murine/human uPA-PAI-1, TNF, or saline by multi-channel flow cytometry, a representative histogram plot and quantitative data are shown (mean±SEM for n=4-7 mice/human blood samples per group; #p<0.05 vs. saline; One-way ANOVA).

(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).
uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils (2021)
Figure 3

Figure 3. Phenotypic and functional properties of uPA-PAI-1-primed neutrophils.

(a) Correlation of uPA or PAI-1 protein expression and neutrophil infiltration in tumors as assessed by ELISA as well as immunohistochemistry and light microscopy in human breast cancer samples (histological grade: G1), representative images (scale bar: 100 μm) and quantitative data are shown.

(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.

(c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).

(d, e) Proliferation of (d) 4T1 breast cancer cells or (e) bEnd.3 microvascular endothelial cells upon exposure to recombinant murine uPA-PAI-1, the uPA-PAI-1 inhibitor WX-340, or primary neutrophils isolated from the peritoneal cavity of WT mice undergoing 6 h of intra-peritoneal stimulation with uPA-PAI-1 with or without addition of a NE inhibitor as assessed by a MTT assay, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. neutrophils; *p<0.05 vs. neutrophils + vehicle / #p<0.05 vs. 2 % FCS medium; One-way ANOVA / t-test).

(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).
uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils (2021)
Figure 4

Figure 4. Effect of WX-340 on experimental breast cancer progression and metastasis formation.

(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).

(b) Intravascular rolling and firm adherence as well as transmigration of neutrophils and classical monocytes (cMOs) to the inflamed perivascular tissue as assessed 6 h after intrascrotal injection of TNF in postcapillary venules of the cremaster muscle of WT mice, quantitative data are shown (mean±SEM for n=6 mice per group; #p<0.05 vs. saline; *p<0.05 vs. drug vehicle; One-way ANOVA).

(c) Expression of uPA or PAI-1 (green) in tumors as assessed in an orthotopic model of 4T1 breast cancer in WT mice by confocal microscopy, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted (scale bar: 100 µm).

(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).

(e) Intravascular rolling and firm adherence of neutrophils as assessed in the tumor microvasculature 14 days after intra-dermal injection of 4T1 cells in the right ear of WT mice treated with WX-340 or vehicle by multi-channel in vivo microscopy, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).

(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).

Expanded view figures
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 1

Figure 1. Changes in NL interactions of Sox6 (a) and Nrp1 (b) in mES cells after activation by TALE-VP64.

A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 2

Figure 2. Local NL detachment caused by gene activation by CRISPRa in human RPE-1 cells.

(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.

(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right. Corresponding DamID traces are shown in Figures 2a-b, 4-5, EV2, EV4.

(d) Color key of domainograms in a-c. Increases in NL interactions (red) are not shown in (c).
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 4

Figure 4. NL interactions and replication timing around activated genes.

A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 5

Figure 5. Putative compensatory movement near the activated MLK4 gene.

Changes in NL interactions after CRISPRa activation of MLK4 in human RPE-1 cells. Visualization of DamID data as in Figure 2a-b.
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 6

Figure 6. Effects of activation of multiple neighboring genes on NL interactions.

(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both.

(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously.

Data information: All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b. See Figures EV2 and EV5 for additional visualization of these DamID data.
Local rewiring of genome-nuclear lamina interactions by transcription (2020)
Figure 7

Figure 7. Increased NL interactions upon allele-specific transcription inactivation in F1 hybrid mES cells.

(a) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc, Morc1 and DppA2 (deletions marked by yellow vertical boxes), and in control cells.

(b) Same as (a), but for the non-mutated Cast alleles.

(c-d) Same design as (a-b), but with only a single mono-allelic deletion of the Morc promoter (vertical yellow box).

(e-f) Effect of PAS insertion on NL interactions of Cobl gene locus. Same design as (a-b) but with insertion of a PAS (located at red triangle and vertical dotted line) that truncates the 129Sv allele of the Cobl transcription unit. 129Sv allele is shown in (e), non-mutated Cast allele in (f).

Data information: Clones with Morc1 locus mutations (each assayed in four independent biological replicates) served collectively as control in (e-f), and the clone with the PAS integration (eight independent biological replicates) served as control in (a-d). Visualization of DamID data in all panels is as in Figure 1.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 1

Figure 1. Myeloid cell lineage-restricted Atx gene deletion accelerated the development of spontaneous gut inflammation in an Il10 deficient condition.

(A) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA: active UC (n=26), active CD (n=34), control (Con) (n=26).

(B) Age- and sex-matched Atx∆ΜΕ/∆ΜΕ;Il10-/- mice (n=9) and Atx+/+;Il10-/- littermates (n=9) were examined for the development of spontaneous colitis. Body weight changes were monitored every other day, starting at the age of 37 days and ending at the age of 6 months. Results are means ± SD. Data were compared by Two-way ANOVA (with treatment and times), followed by the multiple-comparison Bonferroni t test to assess differences between groups.

(C) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).

(D and E) H&E stained section of the mouse colon. Insets are enlarged in the lower panel. Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope (n=14/group) (E).

(F) Histological parameters were quantified with H&E-colon sections (n=10/group) (D) and shown as mean ± SEM.

(G) Survival of Atx∆ΜΕ/∆ΜΕ;Il10-/- (n=17) and Atx+/+;Il10-/- littermates (n=18) was analyzed by the Kaplan-Meier method (Log-rank P=0.0046).

(H) Presented are H&E-stained sections of the small intestine in a 'Swiss-Roll' form. Inset areas are enlarged in the right panel.

Data information: Scale bar indicates 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001 (Mann-Whitney U test in A, E and F, two-way ANOVA in B). The data (B and G) were analyzed with results accumulated from four independent experiments. Presented images are representative.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 2

Figure 2. The integrity of lipid rafts was disrupted in Atx-ko macrophages.

(A) Total cell lysates of peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were subjected to immunoblotting analysis to confirm Atx deletion.

(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).

(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.

(E to G) Lipid rafts from peritoneal macrophages (E, F) or from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.

(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.

Data information: Presented are the representative images of three independent experiments. In the confocal images, more than 95% of the cells exhibited similar results.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 3

Figure 3. Atx deficiency disturbed the formation of the LPS-sensing-TLR4 receptor complex.

(A and B) Peritoneal macrophages (A) or Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.

(C and D) The physical interaction of TLR4-CD14 or TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) or TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 4

Figure 4. The LPS-induced internalization of TLR4 was inhibited in Atx-ko macrophages.

(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 5

Figure 5. Recruitment of TLR4-associated adaptor molecules was inhibited in Atx-ko macrophages.

(A to E) The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) (A, C, E). Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) (B, D). The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 6

Figure 6. TLR4-induced signaling pathways were inhibited in Atx-ko macrophages.

(A to C) The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice (A, B) or Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS [20 ng/mL, 20 min (A, B) or indicated time point (C)].

(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor.

(E to H) The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E), Pam3CSK4 (1 μg/mL) (F), IL-1β (100 ng/mL) (G), or TNFα (25 ng/mL) (H) for the indicated time points.

Data information: The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, regular Akt, β-Actin, or RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 7

Figure 7. LPS-induced cytokine production was reduced in Atx-ko macrophages.

(A and B) Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) or Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.

(C to E) Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.

(F) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).

(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.

Data information: The data (F and G) were analyzed with the results accumulated by 3 independent experiments. All other data are the representative from three independent experiments. **P < 0.01, ***P < 0.001 (Mann-Whitney U test).
Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses (2020)
Figure 8

Figure 8. Innate immune effector function was suppressed in Atx-ko macrophages compare to controls.

(A and B) Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A) and a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).

Scale bars are 5 μm (A) and 50 μm (B), respectively.

(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.

(D and E) After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D). With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).

Data information: All images presented are the representative from at least three independent experiments.

EXPANDED VIEW FIGURE LEGENDS
Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning (2018)
Figure 1

Figure 1. The lack of Tel1 causes a specific hypersensitivity to CPT.

A Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without methyl methanesulfonalte (MMS), hydroxyurea (HU), phleomycin (Phleo) or camptothecin (CPT) at the indicated concentrations.

B Appropriate dilutions of cell cultures were distributed on YEPD plates with or without different concentrations of CPT. Plates were incubated 3 days at 25°C to determine the colony-forming units. Plotted values are the mean values with error bars denoting SD (n = 3).

C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points.

D, E Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT.

F Colony-forming unit-assay as in (B).
Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning (2018)
Figure 3

Figure 3. CPT triggers the hyperactivation of a Mec1-dependent checkpoint in the absence of Tel1.

A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluate DNA contents by flow cytometry (A), nuclear division by fluorescence microscopy (B) and Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C).

D Exponentially growing cell cultures (exp) were treated with CPT (50 μM) for 1 hour, before being released in fresh medium without CPT. Western blot analysis with anti-Rad53 antibodies of protein extracts prepared at the indicated time points.

E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies.