Fig 2: MIF and PAR1 immunostaining in urothelial cells.Representative samples from MIF, PAR1 immunostaining and an overlay showing both and nuclear staining (DAPI; blue). UROtsa cells displayed MIF (A) and PAR1 (B) immunostaining simultaneously in the same cell (C; overlay). However, a number of cells were observed that displayed neither immunostaining (C; arrows) and only showed nuclear staining. Control slides where primary antisera had been omitted (D;E) showed only nuclear staining (F). In rat urothelium, MIF immunostaining was detected in basal and intermediate cells with surface cells displaying weak or no MIF immunofluorescence. PAR1 immunostaining was also observed in rat urothelium, mainly on basal cells but also in some intermediate cells (H) while surface cells showed no PAR1 staining. Overlay of the single staining panels showed that basal cells and some intermediate cells were positive for both MIF and PAR1. Calibration bar = 20 m.

Fig 3: Model of urothelial MIF release mechanisms in vivo.Evidence suggests that substance P elicits MIF release in a process that is dependent on bladder afferent and post-ganglionic activation of muscarinic and adrenergic bladder receptors. Since this release appears is initiated by substance P, it is referred to as “neurogenic” MIF release [9], [10], [24]. In addition, findings from the present experiments show that activation of PAR1 receptors in deep layers of the urothelium can also elicit MIF release. Since this effect occurs in vitro (UROtsa) cells, this pathway appears to be independent of nerve-activation. However, nerve-activation may still contribute, since intravesical lidocaine (to stop all bladder nerve activity) reduced such release in vivo. This alternate pathway for MIF release may be activated by thrombin generated locally as part of the inflammation process or may be activated by proteases found in the urine. Activation of either or both pathways results in MIF release from urothelial cells, MIF binding to cell-surface CD74 in urothelial cells and activating extracellular regulated kinase (ERK) signaling and a pro-inflammatory cytokine cascade. Thus, by standing upstream of such a pro-inflammatory cytokine cascade, MIF maintains or augments bladder inflammation.

Fig 4: Effect of specific microRNA inhibitors on MV-induced inhibition of tumor growth and on modulation of target proteins. (A–D): Tumor growth in severe combined immunodeficiency (SCID) mice injected subcutaneously with 3 × 106 HepG2 and treated weekly with vehicle (?, HepG2) or with 100 µg of MV-HLSC in the presence of anti-miR31 (?) or anti-miR451 (?) or anti-CTR (?). Tumor mass was determined weekly by caliper (A) and by measure of tumor size (B) and weight (C) at sacrifice. (D): Apoptotic cells within the tumors were evaluated by terminal dUTP nickend labeling assay (TUNEL) assay. Results in (B–D) are expressed as mean ± SD of six mice for experimental group. *, p < .05 versus HepG2; §, p < .05 versus anti-CTR+MV-HLSC. (E): Representative micrographs showing the in vivo expression of MDR1, MIF, and RAB14 in HepG2, the downregulation of targets by MV-HLSC (anti-CTR+MV), and the abrogation of downregulation by anti-miR451 treatment (anti-miR451+MV). Treatment with anti-miR451 without MV administration (anti-miR451) did not interfere with protein expression by HepG2. (F): Representative micrographs showing the in vivo expression of E2F-2 in HepG2, its downregulation by MV-HLSC treatment in the presence of anti-CTR inhibitor (anti-CTR+MV), and the abrogation of downregulation by anti-miR31 (anti-miR31+MV). Treatment with anti-miR31 without MV administration (anti-miR31) did not interfere with E2F-2 expression by HepG2. Micrographs are representative of six different experiments. Scale bar = 10 µm. Abbreviations: anti-CTR, negative control miRNA; HLSC, human adult liver stem cell; MV-HLSC, HLSC derived MV; MDR, multidrug resistance protein 1; RAB14, ras-related protein 14; MIF, migration inhibitory factor.

Fig 5: UROtsa cell constitutively express MIF and PAR receptors.Results from RT-PCR experiments showed that UROtsa cells express MIF mRNA (A) and all 4 PAR receptors (B; lanes 1–4 represent PAR1-4 respectively). MIF Western-blotting of UROtsa homogenates (3 representative samples included; C) showed a strong band at approximately 12 kDa corresponding to monomeric MIF (arrow). In addition, 2 distinct MIF bands were observed at higher molecular weight (approximately 80 and 120 kDa) corresponding to MIF binding to protein complexes as described in other systems [24], [25].