Fig 2: SARS-CoV-2’s Spike protein and NRP-1 antagonism reverses SNI-induced mechanical allodynia.Spared nerve injury (SNI) elicited mechanical allodynia 10 days after surgery. (A) Representative immunoblots of NRP-1, total and pY1175 VEGF-R2 levels at presynaptic sites in rat spinal dorsal horn after SNI. Tissues were collected 3 hours (i.e., at peak of anti-allodynia) following intrathecal injection of the receptor binding domain of the Spike protein (2.14 µg/5µl). (B) Bar graph with scatter plots showing the quantification of n= 4 samples as in A (*p<0.05, Kruskal-Wallis test). Paw withdrawal thresholds for SNI rats (male) administered saline (vehicle) or four doses of the receptor binding domain of the Spike protein (0.00214 to 2.14 µg/5µl) intrathecally (i.t.); n = 6-12) (C), 2.14 µg/5µl dose of Spike in female rats, or the NRP-1 inhibitor EG00229 in male rats (2 µg/5µl; n = 6) (E). (D, F, H) Summary of data shown in panels C, E, and G plotted as area under the curve (AUC) for 0-5 hours. P values, versus control, are indicated. Data is shown as mean ± s.e.m. and was analyzed by non-parametric two-way analysis of variance where time was the within subject factor and treatment was the between subject factor (post hoc: Sidak). AUCs were compared by Mann-Whitney test. The experiments were analyzed by an investigator blinded to the treatment. P values of comparisons between treatments are as indicated; for full statistical analyses see Table S1.

Fig 3: Transcript levels of VEGF receptors/co-receptors during in vitro MMT.mRNA levels of VEGF receptors/co-receptors were analyzed by quantitative RT-PCR. The results represent the relative mRNAs expression of VEGF receptors in omentum-derived MCs treated with TGF-ß1 plus IL-1 ß (Omentum T+I) compared with untreated MCs (Omentum NT). The data are depicted as mean value ± SE of omentum samples from 11 healthy donors. (A, B and D) The histograms show a down-regulation in expression of receptors VEGFR-1 (p = 0.005) and VEGFR-2 (p = 0.0005) and up-regulation of co-receptor Nrp-1 (p = 0.004). (C and E) The expression of VEGFR-3 and Nrp-2 did not show significant differences.

Fig 4: Overexpression of NRP1 promotes NSCLC cell migration and invasion. (A) The protein expression levels of Snail, N-cadherin, Vimentin, MMP2 and MMP9 in stable NRP1-overexpressed A549 and H226 cells were detected by western blot analysis. (B) The migratory ability of stable NRP1-overexpressing A549 and H226 cells was investigated by wound healing assays (magnification, x200). (C) The migration and invasion of stable NRP1-overexpressed A549 and H226 cells were investigated by Transwell assays (magnification, x200). Data are shown as the means ± SD. Significantly different from control (PVLX) (**P<0.01 and ***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer.

Fig 5: NRP1 expression correlates with melanoma AJCC stage, tumor thickness and ulceration. (A) Kruskal-Wallis test for differences in NRP1 staining among AJCC stages I–IV. The median is depicted as a horizontal line in each group (n=377, P=0.007). (B) Difference in NRP1 expression among AJCC stages I–IV (n=377, P=0.003, ?2 test). Melanomas in AJCC stages II, III and IV exhibited a higher percentage of high NRP1 expression compared with melanomas in stage I (n=377, P=0.0005, ?2 test). (C) Differences in NRP1 staining between melanoma thickness =2.0 mm and >2.0 mm. The median is depicted as a horizontal line in each group (n=250, P=0.007, t-test). (D) Melanomas >2.0 mm exhibited a higher percentage of high NRP1 expression compared with melanomas =2.0 mm (n=250, P=0.004, ?2 test). (E) Differences in NRP1 staining between melanoma patients with no ulceration and with ulceration. The median is depicted as a horizontal line in each group (n=250, P=0.004, t test). (F) Increased NRP1 expression was correlated with ulceration of melanomas (n=250, P=0.004, ?2 test). NRP1, neuropilin 1; AJCC, American Joint Committee on Cancer.