(D) (Left) Schematic representation of the apoptotic signal induced by Fas-Ab and the antiapoptotic signal induced by Met activation. factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the TMEM2 successful application of aptamer-based receptor agonists. INTRODUCTION The hepatocyte growth factor (HGF) is usually a natural protein that stimulates fundamental cellular functions, such as proliferation, differentiation, and migration (= 3). Statistical significance was examined by two-sided Students test (** 0.01; * 0.05). Agonistic functions and therapeutic potential of the Met agonist aptamer To address whether the aptamer can activate Met signaling in the liver, we evaluated the phosphorylation level of Met by Western MGCD-265 (Glesatinib) blotting 10 min after intravenous injection of the MGCD-265 (Glesatinib) aptamer (Fig. 5B). The administration of the Apt-dimer substantially increased the phosphorylation level of Met and downstream signaling, such as extracellular signalCregulated kinase (ERK) and Akt. These results suggest that a portion of Apt-dimer is usually delivered to the liver in a functional form by avoiding nuclease digestion. Indeed, 3% dose per gram of tissue of the Apt-dimer was detected in the liver by quantitative real-time polymerase chain reaction (PCR)Cbased analysis (fig. S7). Because Met was expressed mainly in hepatocytes in the liver parenchyma (fig. S4), these data strongly suggest the functionality MGCD-265 (Glesatinib) of the Apt-dimer to activate the HGF/Met-signaling cascade in hepatocytes. Immunohistochemical analysis performed using MGCD-265 (Glesatinib) a phospho-MetCspecific antibody also revealed the aptamer-induced Met activation throughout the liver tissues, including hepatocytes (Fig. 5C and fig. S8). These results are consistent with the direct observations of Apt-dimer distribution, which showed Apt-dimer leakage to the liver parenchyma (Figs. 2D and ?and3A).3A). In contrast, the G4 mutant did not induce Met phosphorylation, as assessed using Western blotting and immunohistochemical analysis, supporting the contention that Met phosphorylation was induced in a manner MGCD-265 (Glesatinib) that was specific to the Apt-dimer. Open in a separate windows Fig. 5 Agonistic functions and therapeutic potential of the HGF-mimic aptamer.(A) Schematic representation of the experimental outlines. (B) Immunoblotting analysis of Met activation in mouse livers after intravenous injection of recombinant human HGF (rhHGF) or oligonucleotides. The liver was excised 10 min after the intravenous injection of rhHGF (1 g) or oligonucleotides (1 to 10 nmol). (C) Immunohistochemistry of Met activation in the mouse liver after intravenous injection of the oligonucleotides. The liver was excised 10 min after the intravenous injection of oligonucleotides (10 nmol). (D) (Left) Schematic representation of the apoptotic signal induced by Fas-Ab and the antiapoptotic signal induced by Met activation. (Right) Effect of Apt-dimer administration on a fulminant hepatitis mouse model. An antiCFas-Ab (2 ng) was co-injected with vehicle control (= 10), Apt-dimer (10 nmol, = 9), or G4 mutant (10 nmol, = 9) intravenously. The oligonucleotide (10 nmol) was administered 1.5 hours later. The average activity of glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), and lactate dehydrogenase (LDH) is usually shown with error bars (SD). Statistical significance was examined by one-way analysis of variance (ANOVA) followed by Tukeys post hoc test (*** 0.001; ** 0.01; ns, not significant). Last, we evaluated the therapeutic potential of the Apt-dimer using a fulminant hepatitis mouse model. In the present study, an agonistic Fas antibody (Fas-Ab) with proapoptotic function was systemically injected into mice to induce massive apoptosis of hepatocytes, which leads to liver failure (Fig. 5D, left) (for 20 min at 4C, and then the supernatants were recovered. The total protein concentration of each cell lysate was adjusted to the same value according to the result obtained by bicinchoninic acid (BCA) assay. The cell lysates were separated by SDS-PAGE and then transferred to the polyvinylidene difluoride (PVDF) membrane. The membrane was reacted with the following primary antibodies at 4C overnight: antiCphospho-Met Y1234/Y1235 (1:2000; 3077, Cell Signaling Technology), anti-Met (1:2000; 3127, Cell.
- 4 EGFR-targeted nanobodyCPS conjugates localize in tumors specifically
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- FITC- and Cy3-conjugated supplementary antibodies were utilized to visualize the tagged ORF65 and TGN46 protein, respectively
- Dedication of CIV-DP To measure CIV-DP concentrations, a sandwich ELISA was used
- (D) (Left) Schematic representation of the apoptotic signal induced by Fas-Ab and the antiapoptotic signal induced by Met activation