Fig 1: High NTS and NTSR1 expression indicated poor prognosis in glioma patients. A, Scatter grams of NTS expression levels, as well as ProNTS and NTSR1 expression quantification of western blot in 30 glioma specimens with different histological grades; the p values are indicated. B, Box plot of NTS expression levels in different pathological types of glioma and different grades in the Sun dataset; the p values compared to the astrocytoma group are indicated. C, Kaplan–Meier analysis of overall survival according to NTS and NTSR1 expression for the Gravedeel dataset; p values were determined using the log-rank test and were indicated.
Fig 2: NTS/NTSR1 promoted the proliferation of glioma cells. A, The expression of NTSR1 in GL261 and U87 glioma cells were detected by immunofluorescence staining. B, CCK8 were used to test the effect of NTS, SR48692 and NTSR1-siRNA on the proliferation of GL261 and U87 glioma cells. C, Immunofluorescence staining showed BrdU-positive GL261 and U87 glioma cells in the different groups after BrdU incorporation. D, The histogram of BrdU incorporation experiments. *p < 0.01 vs. the control group using a two-tailed t test, **p < 0.01 vs. the control + sc-siRNA group using a two-tailed t test, # p < 0.01 vs. the NTS + sc-siRNA group using a two-tailed t test. A, B Merged images; the nuclei were counterstained with DAPI and are shown in blue. Laser confocal scanning microscopy, scale bar = 20 μm.
Fig 3: NTS induced the activation of Erk1/2 through NTSR1 in GL261 glioma cells. A, GL261 glioma cells were stimulated with 50 nM NTS for the indicated times. B, Glioma cells were stimulated with NTS at indicated concentrations for 5 min. C, GL261 glioma cells were stimulated with NTS for 5 min in the absence or presence of 5 μM SR48692 or 2 ng/ml NTS-NA. *p < 0.01 vs. the control group, # p < 0.01 vs. the 100 nM NTS-stimulated group. D, GL261 glioma cells were stimulated with NTS for the indicated times in the presence of Sc-siRNA or NTSR1-siRNA. *p < 0.01 vs. the 100 nM NTS-stimulated group in the presence of Sc-siRNA for 2 min, # p < 0.01 vs. the 100 nM NTS-stimulated group in the presence of Sc-siRNA for 5 min. The cell lysates were evaluated by western blot analysis using antibodies against phospho-Erk1/2. The results were standardized to the total levels of Erk2 and are expressed as the mean ± SEM from at least three independent experiments. E and F, The percentages of BrdU-positive cells and invasived cells in the presence of SR48692 and the MEK1/2-selective inhibitor U0126. *p < 0.01 vs. the control group, # p < 0.01 vs. the control group.
Fig 4: NTS/NTSR1 boosted the migration capacity and invasiveness of glioma cells. A, Transwell invasion assay showed that the ability of GL261 and U87 glioma cells to invade across the matrigel and membrane. B, The proportion of invasive GL261 and U87 glioma cells in all the experimental groups in the transwell experiments. *p < 0.01 vs. the control group using a two-tailed t test, & p < 0.01 vs. the NTS group using a two-tailed t test. # p < 0.01 vs. the NTS + sc-siRNA group using a two-tailed t test. C, Illustrations of the scratch wounds inflicted by a pipette tip. After 36 hours, the scratch wounds were recolonized by GL261 cells cultured in low-serum medium (0.1% FCS). D, The percentage of wound closure by the cells was quantified in the different groups at 36 hours and 72 hours. && p < 0.01 vs. the NTS group using a two-tailed t test. **p < 0.01 vs. the control group using a two-tailed t test, ## p < 0.01 vs. the NTS + sc-siRNA group using a two-tailed t test.
Fig 5: NTSR1-selective antagonist SR48692 inhibited glioma progression in vivo . A, General observation showed that brain swelling was present in the mice of the control group (dotted black circle) after 10 days of intracranial incubation, scale bar = 1 cm. Glioma progressed and invaded the contralateral tissue during the early stages of tumor formation in the control group (black arrows). H.E. staining. Bar = 1 mm. B, The MRI detection revealed the growth of intracranial tumors in the 10 mg/kg SR48692 treatment group and the control group. Scale bar = 5 mm. The Tv curve was also showed. # p < 0.01 vs. the control group. Kaplan-Meier survival curve of the glioma-bearing mice in the control group and the SR48692 treatment groups. p values vs. the control group are indicated. The MRS analyses in voxels placed in tumor core (blue square) and peritumoral regions (red square) showed the Cho/NAA ratios and Cho/Cr ratios between the SR48692 treatment group and the control group. The P values vs. the control group are indicated. C, H.E. staining showed the margin of gliomas (black dotted line) in the control group and the 5 mg/kg SR48692 treatment group Scale bar = 100 μm. D, NTS immunoreactivity was observed in syngeneic xenografts using IHC staining. The images with a larger magnification in the corner of figures. Scale bar = 100 μm.
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