Fig 1: Final scheme of potential EMT regulation by uPA/uPAR cooperation in Neuro2a cells. (a) uPAR functions as a uPA “trap.” When uPAR is expressed on the cell surface, binding of uPA to uPAR leads to rapid internalization of the ternary uPA/uPAR/PAI‐1 complex via the LRP‐1 receptor with subsequent lysosomal degradation of uPA and PAI‐1; uPAR and LRP‐1 recycle to the plasma membrane. (b) In the absence of uPAR, uPA is translocated to the nucleus, where it may be involved in the regulation of the activity of transcription factors Snai1 and NF‐κB leading to increased expression of IL‐6, N‐cadherin, and α‐SMA and resulting in epithelial to mesenchymal transition in Neuro2a cells. α‐SMA, α‐smooth muscle actin; EMT, epithelial–mesenchymal transition; IL‐6, interleukin‐6; LRP‐1, LDL receptor‐related protein 1; NF‐κB, nuclear factor κB; uPA, urokinase‐type plasminogen activator; uPAR, urokinase‐type plasminogen activator receptor
Fig 2: Downregulation of uPAR in Neuro2a leads to uPA and NF‐κB translocation into the nucleus. (a) RT‐PCR analysis of uPA expression level. The mRNA level of uPA was normalized to β‐actin expression as a housekeeping gene; the normalization was done assuming the mean level of transcript in WT cells to be 1. The bar graph represents the mean relative expression level of at least three biological repeats ± SEM. *p < .05, **p < .01, ***p < .001 compared with WT cells (unequal variances, Welch's ANOVA, Tamhane's T2 post hoc tests). (b) RT‐PCR analysis was carried out to evaluate the expression of uPAR in control Neuro2a cells (WT) and clones after re‐expression of uPAR (Re#3, Re#6, and Re#30). Data were normalized to β‐actin expression as a housekeeping gene. Results are representative of three independent experiments. (c) Accumulation of uPA and NF‐κB in the nuclear fractions of Neuro2a cells with different levels of uPAR expression using western blot analysis assay. Results are representative of three independent experiments. (d) Double immunofluorescent staining of Neuro2a cells with antibodies against uPA (red fluorescence) and LAMP1‐lysosomal marker (green fluorescence). Nuclei were counterstained with DAPI (blue fluorescence). The results of at least three independent experiments are presented. The distribution of pixel intensity along the red arrow passing through the region of the nucleus is shown on the left side of each image. (e) Graph representing the results of the fluorescence intensity analysis of uPA content in the nuclei of Neuro2a cells. Results are representative of three independent experiments. (f) Confocal microscopy of Neuro2a cells 30 min and 6 hr after administration of FITC‐conjugated uPA (green fluorescence) to the cell culture medium. Immunofluorescent staining using antibodies against LAMP1 (red fluorescence) was carried out under permeabilizing conditions, nuclei were counterstained with DAPI (blue fluorescence). The results of at least three independent experiments are presented. (g) The same experiment as in (f) after 24 hr incubation with uPA‐FITC; channels are presented separately or merged (DAPI blue fluorescence, uPA‐FITC green fluorescence, LAMP1 red fluorescence). The results of at least three independent experiments are presented. ANOVA, analysis of variance; DAPI, 4′,6‐diamidino‐2‐phenylindole; FITC, fluorescein isothiocyanate; LAMP1, lysosomal‐associated membrane protein; NF‐κB, nuclear factor κB; mRNA, messenger RNA; RT‐PCR, real‐time polymerase chain reaction; SEM, standard error of mean; uPA, urokinase‐type plasminogen activator; uPAR, urokinase‐type plasminogen activator receptor; WT, wild‐type
Fig 3: Expression of uPAR affects cell morphology in Neuro2a cells. (a) Neuro2a cells with different levels of uPAR expression were plated in 24‐well plates at a concentration of 1 × 104/well. 48 hr after the cells were monitored using a light microscope. Results are representative of three independent experiments. Bar 75 µm. (b) Results of cell size (cell area) measurement; minimal cell size was limited to 50 μm2 to avoid measuring too small dots. (c) Control Neuro2a cells (WT) and cells transfected with shRNA to suppress uPAR (Neuro2a‐shuPAR) were plated onto 24‐well plates at a concentration of 0.5 × 104/well. 48 hr later, cells have been monitored using light microscopy. The silencing of uPAR in Neuro2a cells leads to the emergence of atypical cells of large size. Results are representative of three independent experiments. Bar 50 µm. (d) Results of cell size (cell area) measurement; minimal cell size was limited to 50 μm2 to avoid measuring unrelated dots. (e) Neuro2a cells with different uPAR expression were seeded into cell culture plates in the presence of 50 nM uPA or BSA as a control. 24 hr later, cells were analyzed using light microscopy. Bar 250 µm. Results are representative of three independent experiments. (f) Graph showing the number of colony‐forming units (CFU) calculated at least in three fields of view. The cell colony‐forming ability of Neuro2a depends on the uPAR expression. Data are presented as mean ± SEM, *p < .05 compared with WT, **p < .01 compared with WT, ***p < .001 compared with WT, ****p < .0001 compared with WT (ANOVA, Tukey post hoc test). WT: control Neuro2a cells; Neuro2a‐uPAR: uPAR‐overexpressing Neuro2a cells; #3, #6, and #30: uPAR‐deficient clones of Neuro2a cells. ANOVA, analysis of variance; BSA, bovine serum albumin; SEM, standard error of mean; shRNA, short hairpin RNA; uPA, urokinase‐type plasminogen activator; uPAR, urokinase‐type plasminogen activator receptor; WT, wild‐type;
Fig 4: uPAR downregulation decreases Neuro2a cell adhesion and increases Neuro2a cell migration in wound scratch assay. (a) Neuro2a cells with different levels of uPAR expression were seeded into cell culture plates and the medium with non‐adherent cells was replaced 30 min, 1 or 2 hr later. Neuro2a cell adhesion was analyzed using light microscopy. Bar 250 µm. Results are representative of three independent experiments. (b) Graph showing the mean number of attached cells calculated at least in three fields of view in three different wells ± SEM, *p < .05 compared with WT cells (two‐way ANOVA, Tukey post hoc test). (c) Neuro2a cells were allowed to form a confluent monolayer. A scratch was created in a straight line with a p1000 pipet tip across the monolayer of cells. During 24 hr cells were cultured in the presence of 50 nM of uPA, 10 μM BC11 (uPA inhibitor) or BSA as a control. Green areas indicate cell‐free zones as determined using the MRI Wound Healing Tool of ImageJ. Results are representative of three independent experiments. Bar 250 µm. (d) Statistical analysis of the wound scratch closure monitored over time in the presence of BSA (blue columns), uPA (red columns), or BC11 (green columns). Data are presented as the mean residual wound area at 24 hr as a percentage of the original wound area at 0 hr ± SEM. *p < .05, **p < .01, ***p < .001, ****p < .0001 all compared with the respective value in WT BSA, WT uPA, or WT BC11 (two‐way ANOVA, Tukey post hoc test). The factor of different agents (BSA, uPA, or BC11) administrated to the cell medium was statistically nonsignificant (p > .05, two‐way ANOVA). WT: control Neuro2a cells; Neuro2a‐uPAR: uPAR‐overexpressing Neuro2a cells; #3 and #6: uPAR‐deficient clones of Neuro2a cells. ANOVA, analysis of variance; BSA, bovine serum albumin; MRI, magnetic resonance imaging; uPA, urokinase‐type plasminogen activator; uPAR, urokinase‐type plasminogen activator receptor; SEM, standard error of mean; WT, wild‐type
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