EMBO/soda-nel huggingface dataset:
Gel-like inclusions of C-terminal fragments of TDP-43 sequester stalled proteasomes in neurons (2022)
Figure 1

Figure 1: TDP-25 forms amorphous inclusions enriched in proteasomes.

A: Primary rat hippocampal neurons were transduced with constructs expressing GFP-tagged TDP-25 (amino acids 220-414 of TDP-43) on day 5 in vitro and cultured for 8 additional days (DIV5+8). Wild-type or variant containing 8 ALS-causing mutations. Immunofluorescence shows disease-related phosphorylation at serine 403/404. Counterstain to label the neuronal cytoskeleton (MAP2) and nuclei (DAPI). Scale bar = 10 µm.

B: Hippocampal neurons transduced with GFP-TDP-25 constructs (DIV5+8) or GFP-TDP-43 (DIV5+4 due to higher toxicity) were sequentially extracted with RIPA buffer followed by 2% SDS buffer and analyzed via immuoblotting. Left panel shows representative immunoblot for GFP and loading control Calnexin. Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract. Barplots showing mean ± SD from n=3 independent experiments. TDP-43 (0.039 ± 0.029, mean ± CI) vs TDP-25 wt (1.22 ± 0.296) vs TDP-25 mut (0.928 ± 0.564): F(2,6)=51.4, ***p=0.000168, η²=0.94. TDP-43 vs TDP-25 wt: ***p=1.67*10-4, TDP-43 vs TDP-25 mut: p=7.983*10-4, TDP-25 wt vs TDP-25 mut: p=0.1182, One-way ANOVA with Tukey's post-hoc test.

C: Tomographic slice of an aggregate within a GFP-TDP-25 wild-type transduced neuron (DIV5+8). Colored boxes show a series of higher magnification tomographic slices of representative protein complexes detected in the tomogram. Green boxes show side views of single-capped, yellow boxes show smaller ring-like structures of 26S proteasomes. Red dotted line segments aggregate area. Scale bar = 200 nm.

D: 3D rendering of the aggregate shown in (C). Amorphous aggregate material is labeled in red, proteasomes in violet, ribosomes in yellow, TRiC/CCT chaperones in green, mitochondria (Mt) in white, ER in pink and other vesicles (V) in light yellow. The irregular aggregate structures were approximately segmented using a threshold-based approach for visualization purposes. Average structures of proteasomes, ribosomes and TRiC/CCT were pasted at the locations and orientations determined by template matching. See also Movie EV1.

E: Subtomogram averaging of macromolecules in GFP-TDP-25 inclusions reveals the proteasome structure at ~20 Å resolution (see Fig. EV1). The positions of Rpn5/PSMD11 and Rpn6/PSDM12 are indicated. Prominent extra densities in the substrate binding region are colored in pink in the 3D rendering.

F: Classification based on the conformation of the regulatory particle in TDP-25 inclusions compared to non-transduced control neurons (Asano et al., 2015) and poly-GA inclusions (Guo et al., 2018). GS = Ground State, SPS = Substrate Processing State. Due to the uncertainties inherent to the classification procedure, it is possible that a small fraction of particles adopted other conformations.
Gel-like inclusions of C-terminal fragments of TDP-43 sequester stalled proteasomes in neurons (2022)
Figure 2

Figure 2: TDP-25 inclusions are neither liquid-like nor solid-like.

(A-D) Primary rat hippocampal neurons were transduced with GFP-TDP-25 variants, GFP-NPM1 or Htt97Q-GFP and analyzed by fluorescence recovery after photobleaching (FRAP) at DIV5+8, except Htt97Q-GFP which was analyzed at DIV5+6 to avoid excessive toxicity. Example images with indicated bleach regions (dashed circles) are shown (A, C). Scale bar = 1.5 µm. Resulting normalized FRAP curves (relative fluorescence intensity over time) with values representing means ± SD are shown in (B, D). (B) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments. GFP-NPM1 (0.982 ± 0.019, mean ± CI, n=37 cells) vs GFP-TDP-25 wt (0.097 ± 0.013, n=35 cells) vs GFP-TDP-25 mut (0.085 ± 0.006, n=38 cells): H(1)=74.134, df=2, ***p<2.2*10-16, η²[H]=0.674, Kruskal-Wallis Test. GFP-NPM1 vs GFP-TDP-25 wt: ***p<2*10-16, GFP-NPM1 vs GFP-TDP-25 mut: p<2*10-16, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.11, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. In (D), the recovery fraction averaged at >20 min timepoints was compared from four independent experiments. Htt97Q-GFP (0.094 ± 0.017, n=25 cells) vs GFP-TDP-25 wt (0.312 ± 0.041, n=23 cells) vs GFP-TDP-25 mut (0.271 ± 0.029, n=24 cells): H(1)=46.376, df=2, ***p=8.501*10-11, η²[H]=0.643, Kruskal-Wallis Test. Htt97Q-GFP vs GFP-TDP-25 wt ***p=1.9*10-11, Htt97Q-GFP vs GFP-TDP-25 mut ***p=4.3*10-12, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.12, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.
The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation (2020)
Figure 1

Figure 1. hMENA isoform expression in CAFs

A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.

B. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71 and cancer associated fibroblasts

(n=10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENA∆v6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left.

C. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer associated fibroblasts obtained from NSCLC tissues (n=4) and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENA∆v6 expression.

Data information: Quantified data, are represented as fold change of hMENA∆v6/TUBULIN ratio with respect to control P-NF and L-NF (set as 1).
The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation (2020)
Figure 2

Figure 2. hMENA expression in tumor and stroma of PDAC and NSCLC tissues

A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).

B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).

C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.

D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05).
The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation (2020)
Figure 3

Figure 3. hMENA/hMENA∆v6 regulate pro-tumor CAF functional activity

A. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.

B. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.
The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation (2020)
Figure 6

Figure 6. hMENA/hMENA∆v6 silencing inhibits AXL expression and activity in cancer cells

A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.

B. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001.

C. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001.

D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.

E. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01.

F. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 1

DDX3 expression and knockdown phenotype in lung cancer cell lines and in lung cancer patient samples

A Immunoblot of DDX3 expression in lung cancer cell lines.

B, C Colony-forming assays in H1299 (B) and A549 (C) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control. Corresponding immunoblots displaying knockdown levels of DDX3. Mean from 3 replicates with SD.

D Proliferation of A549 and H1299 cells after knockdown of DDX3. Mean from 3 replicates with SD. (A549 P = 0.011, H1299 P = 0.014; exponential curve fit, extra sum of squares F-test).

E β-galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color.

F Expression of DDX3 by immunohistochemistry in normal lung tissue.

G DDX3 expression in squamous cell carcinoma.

H DDX3 expression in adenocarcinoma.

I DDX3 expression in small cell carcinoma.

J Expression of DDX3 in different histological types of lung cancer. All data sets were compared against each other (chi-square test, P = 0.481).

K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan–Meier curve and log-rank test, P = 0.016).

Data information: Scale bars: 25 μm.

Source data are available online for this figure.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 2

Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell lines

A Chemical structure of RK-33.

B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.

C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon.

D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33.

F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.

G Schematic representation of helicase assay.

H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4–6).

I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.

J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.

Source data are available online for this figure.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 6

RK-33 induces radiosensitization in preclinical mouse models of lung cancer

Schematic describing the Twist1/KrasG12D-inducible mouse model.

Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.

Treatment schedule for mice receiving hypofractionated radiation (SABR). Stars are intraperitoneal (i.p.) injections with RK-33.

Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&E staining of lung sections (lower panel). Scale bar is 250 μm.

Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.

An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.

Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.

Treatment schedule for mice receiving fractionated radiation in 10 fractions. Stars indicate i.p. injections with RK-33. Downward lightning bolts indicate 3-Gy radiation fractions.

Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.

Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 7

Effect of DDX3 knockdown and RK-33 on cell cycle progression and apoptosis

A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.

B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.

D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.

F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.

Source data are available online for this figure.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 8

Effect of RK-33 on Wnt signaling via DDX3

A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.

B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.

C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.

D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.

F–I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.

J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT–PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.

Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.

Source data are available online for this figure.
Targeting DDX3 with a small molecule inhibitor for lung cancer therapy. (2015)
Figure 9

Effect of RK-33 on radiation-induced DNA damage

A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.

B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.

C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.

D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.

E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.

F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.

Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.

Source data are available online for this figure.
uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils (2021)
Figure 1

Figure 1. Effect of uPA-PAI-1 heteromerization on leukocyte trafficking.

(a) Trafficking of neutrophils (N), classical monocytes (cMOs), non-classical monocytes (ncMOs), B lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes to the peritoneal cavity as assessed 6 h after intraperitoneal stimulation with recombinant murine uPA, tPA, PAI-1, uPA-PAI-1, or tPA-PAI-1 in WT mice by multi-channel flow cytometry. The gating strategy and quantitative data are shown (mean±SEM for n=5 mice per group; #p<0.05 vs. saline; One-way ANOVA).

(b) Neutrophil and cMO trafficking to the peritoneal cavity elicited by recombinant murine uPA-PAI-1 as assessed in WT mice receiving neutrophil-depleting anti-Ly-6G mABs or isotype control ABs, quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. isotype; One-way ANOVA).

(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).