Fig 2: Pazopanib reduced the expression of NGF and VEGFA in sensory neurons (DRG), which were associated with OA pain. Innervating lumbar DRG (L3-L5) were assessed for expression of VEGFA and NGF in sensory neurons (using NeuN as a neuron marker, red) by dual immunofluorescence (IF), arrows indicate DRG neurons positive for VEGFA (green) or NGF (green) (A). IF assays were performed in histological sections of DRG tissues in mice at 12 weeks post-partial medial meniscectomy (PMM). IA injection of pazopanib (Paz), vandetanib (Van) or vehicle (Veh, 5% DMSO in PBS) was done at week 1 (Gp1, inflammatory pain stage), week 4 (Gp2, early OA stage) or week 8 (Gp3, advanced OA stage) after PMM, twice per week for 12 weeks, and the effect of drugs or vehicle on the expression of VEGFA and NGF in the DRG was determined. Quantitative analysis showed that VEGFA and NGF expression was significantly increased after PMM (n=3) (B-G). Statistical analysis was conducted using one-way ANOVA followed by the Tukey-Kramer test. *p<0.05, **p<0.01, ***p<0.001 for comparisons between groups with or without drug treatments in mice with PMM. Scale bars: 100 μm.

Fig 3: Pazopanib, not vandetanib, suppressed the number of peripheral nerve fibers sprouting in the synovium-the role of VEGFR1 in sensory nerve distribution in synovium. Immunofluorescence (IF) assays were performed in histological sections of cartilage and synovial tissues in mice at 12 weeks post-partial medial meniscectomy (PMM). IA administration of drugs (Paz, Van) or vehicle (Veh, 5% DMSO in PBS) commenced at week 1 (Gp1, inflammatory pain stage), week 4 (Gp2, early OA stage) or week 8 (Gp3, advanced OA stage) after PMM twice per week for 12 weeks; and the effect of the drugs or vehicle on the expression of VEGFA (green), NGF (green) and PGP9.5 (green) in cartilage and/or synovium was examined by IF microscopy (A). Quantitative analysis showed that VEGFA, NGF and PGP9.5 expressions were significantly increased after PMM (n=3) (B-M). Statistical analysis was conducted using one-way ANOVA followed by the Tukey-Kramer test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 are for comparisons between groups with or without drug treatments in mice with PMM. 4′,6-diamidino-2-phenylindole (DAPI) stains nuclei blue. Scale bars: 100 μm. FI, fluorescence intensity; a.u., arbitrary unit.

Fig 4: Pazopanib reduces spinal NF-kB-glial axis activation. Spinal cords (L3-L5) were assessed for the expression of pNF-κB and VEGFA and the reactivity of astrocytes and microglia using double immunofluorescence (IF). IF assays were performed in histological sections of spinal cord tissues in mice at 12 weeks post-partial medial meniscectomy (PMM). IA injection of pazopanib (Paz), vandetanib (Van) or vehicle (Veh, 5% DMSO in PBS) was performed at week 1 (Gp1, inflammatory pain stage), week 4 (Gp2, early OA stage) or week 8 (Gp3, advanced OA stage) after PMM, twice per week for 12 weeks, and the effect of pazopanib, vandetanib or vehicle on the expression of pNF-κB and VEGFA and the reactivity of astrocytes and microglia in the lumbar spinal dorsal horns were observed; the expression of GFAP (an astrocyte marker, red), IBA1 (a microglia marker, green), pNF-κB (red) and VEGFA (green) was examined by IF microscopy (A). Quantitative analysis demonstrated that the expression of GFAP, IBA1, VEGFA and NF-κB activation were significantly increased after PMM (n=3) (B-M). Statistical analysis was conducted using one-way ANOVA followed by the Tukey-Kramer test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 comparisons between groups with or without pazopanib or vandetanib treatment in mice that underwent PMM. 4′,6-diamidino-2-phenylindole (DAPI) stains nuclei blue. Scale bars: 100 μm.

Fig 5: METTL3 and LINC00662 promoted the proliferation and migration of CRC cells in zebrafish. A Cell proliferation and migration assays in zebrafish after interference with si-NC, si-LINC00662 3#, si-METTL3 3# and si-LINC00662 3#+ pc DNA METTL3 in HCT116 cell. The images of proliferation and migration were taken by Olympus fluorescence microscope IX53 (Olympus, Center Valley, USA), and fluorescence area was calculated by Image J. B Fluorescence area of HCT116 cell proliferation and migration was quantified by Image J. C Fluorescence area of HT29 cell proliferation and migration was quantified by Image J. D Cell proliferation and migration assays in zebrafish after interference with si-NC, si-LINC00662 3#, si-METTL3 3# and si-LINC00662 3#+ pc DNA METTL3 in HT 29 cell. The images of proliferation and migration were taken by Olympus fluorescence microscope IX53 (Olympus, Center Valley, USA), and fluorescence area was calculated by Image J. E Mechanism diagram of METTL3 dual regulation of LINC00662 and VEGFA RNAs stability to promote angiogenesis in CRC