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KIF11 deficiency alters MAPK and AKT signaling pathways and reduces expression of lymphatic cell identity and cell-cell junction markers. (A) Human phospho-kinase array showing changes in phosphorylation levels of 43 kinases. Dots have been magnified for selected kinases with decreased (in blue) or increased (in red) phosphorylation. (B) Network pathway analysis using STRING and Cytoscape reveals known interactions and the directionality of the phosphorylation change in siRNA KIF11-treated cells (blue, decreased; red, increased). (C) Effect of ispinesib treatment (50 nM) on AKT and MAPK phosphorylation in LECs over time after stimulation with VEGFC (100 ng/mL). DMSO (vehicle) as control. Phosphorylation was analyzed with anti-pAKT S473 and anti-p44/42 MAPK Thr202/Tyr204 (upper panels) and levels of AKT and MAPK protein expression with anti-AKT and MAPK antibodies (lower panels). Molecular mass markers (in kDa) are indicated on the left. Blots are quantified on the right. *P < 0.05, **P < 0.01, ns, nonsignificant. Two-tailed unpaired Student’s t test. (D) FLT4 (VEGFR3), PROX1, PDPN, FOXC2, LYVE1, TJP1 (ZO-1), CLDN5, and CDH5 gene expression related to GAPDH in LECs treated with siRNA 6 KIF11 for 48 and 72 hours analyzed by qPCR (n = 3–4 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. (E) Western blot analysis and quantification of PROX1 and VEGFR3 expression in LECs treated with siRNA KIF11 for 48 and 72 hours. GAPDH was used as control. **P < 0.01, ***P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. (C–E) Bars represent mean relative expression ± SEM. (C and E) One representative image is shown from n = 3–4 experiments.
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