Fig 1: NEK9 was increased in primary GC and was correlated with cancer metastases and overall survival periods in patients with GC. A, NEK9 was increased in 2 cohorts of GC patients from Oncomine database. B-D, NEK9 was associated with advanced tumor stage (B, data from TCGA) and reduced overall survival periods (C, data from TCGA. D, data from Kaplan-Meier plotter). E-F, NEK9 was examined in multiple tissue microarrays by IHC and it was increased in primary GC, lymph node metastases and distant metastases. ***P < 0.001. G. An increase in NEK9 was correlated with poor prognosis of patients with GC.
Fig 2: NEK9 promoted cell motility by directly phosphorylating ARHGEF2. A, The screening strategy of potential targets of NEK9. B-C, The total phosphorylation levels on serine, threonine and tyrosine of ARHGAP35 (B) and ARHGEF2 (C) were examined. D-F, The direct interaction between NEK9 and ARHGEF2 was validated by pulldown assay (D) and IP (E), and their colocalization was confirmed by immunofluorescence (F). G, NEK9 was found to directly phosphorylate ARHGEF2 by in vitro kinase assay. ***P < 0.001. H. The function of NEK9 and ARHGEF2 on cell motility was attenuated by mutations on all potential targeted serine residues. **P < 0.01, ***P < 0.001.
Fig 3: Activation of STAT3 by IL-6 directly transcriptionally suppressed miR-520f-3p. A, IL-6 increased cell migration and invasion. **P < 0.01. B, IL-6 led to increased NEK9 expression through STAT3 and miR-520f-3p. C, IL-6 suppressed miR-520f-3p while knockdown of STAT3 increased miR-520f-3p. ***P < 0.001. D, Serially truncated and mutated miR-520f-3p promoter constructs were cloned and transfected into cells. The relative luciferase activities were determined after IL-6 stimulation. **P < 0.01. E, Selective mutation (left panel) analyses identified STAT3-responsive regions in the miR-520f-3p promoter (right panel). **P < 0.01. F, ChIP assay demonstrated the direct binding of STAT3 to the miR-520f-3p promoter (left panel), and the ChIP products were validated by RT-PCR (right panel). **P < 0.01.
Fig 4: NEK9 regulated cell motility in vitro and in vivo. A-B, NEK9 was examined in multiple GC cells (A). Cell models with stable NEK9 transfection were established and validated by western blotting and RT-PCR (B). C-D, NEK9 promoted cell migration, invasion and wound healing process (D), while its knockdown inhibited cell movement (D). *P < 0.05, **P < 0.01. E, Ectopic NEK9 promoted cancer metastasis in vivo, and knockdown of NEK9 suppressed cancer metastasis. *P < 0.05, **P < 0.01. F-G, Ectopic NEK9 promoted the activation of RhoA, manifested as increased GTP-RhoA. Inhibition of NEK9 led to a decrease in GTP-RhoA.
Fig 5: S691, S952 and S956 were the direct and functional serine residues targeted by NEK9. A-C, The phosphorylation on target serine residue was detected (A) and their roles in regulating cell motility was examined (B). The serine residues that met the requirements from A and B were listed as potential targeted serine targeted by NEK9 (C). ***P < 0.001. D, The phosphorylation on target serine residue of ARHGEF2 was increased at the presence of NEK9. E-F, NEK9 enhanced the effects of ARHGEF2 with wide-type S691, S952 and S956 on RhoA activation (E) and cell motility (F).
Supplier Page from Abcam for Recombinant human NEK9 protein