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. 2021 Jan 12;11:644. doi: 10.1038/s41598-020-80060-3

Figure 5.

Figure 5

EPHA10 is required for EFNA4-induced cell migration, sphere formation, and expression of NANOG and OCT4. (A) EPHA2 and (D) EPHA10 levels in OEC-M1 cells expressing EPHA2 or EPHA10 shRNA, respectively, or the corresponding controls (pLKO-GFP) were determined by immunoblot. Protein levels were normalized to an internal control (α-tubulin). Relative ratios were determined by dividing the level of the EPHA2 or (D) EPHA10 in each expression variant by that in the pLKO-GFP vector-expressing cells. (B) Representative data show the relative migration potential of OEC-M1 pLKO-GFP, EPHA2 sh4, and EPHA2 sh5 cells or (E) pLKO-GFP, EPHA10 sh3, and EPHA10 sh5 cells treated with 0.1 μg/ml EFNA4-Fc or 0.1 μg/ml IgG control. Upper: Representative images of migrated cells. Scale bars, 100 μm. Lower: The relative migration activity as determined by normalizing the mean number of migrated cells per field of the knockdown cells treated with EFNA4-Fc (n = 10) to that of control cells (n = 10). (C) The tumorspheres in OEC-M1 pLKO-GFP, EPHA2 sh4, and EPHA2 sh5 cells or (F) pLKO-GFP, EPHA10 sh3, and EPHA10 sh5 cells treated with 0.1 μg/ml EFNA4-Fc or 0.1 μg/ml IgG control were assessed in sphere culture. Relative sphere formation activity was determined by normalizing the mean number of spheres per well of the knockdown cells treated with EFNA4-Fc (n = 2) to that of the control cells (n = 2). (G) Relative levels of NANOG, OCT4, and SNAIL mRNA in OEC-M1 pLKO-GFP, EPHA10 sh3, and EPHA10 sh5 cells treated with EFNA4-Fc or IgG were measured by qRT-PCR and normalized to β-actin (internal control). For each gene, the relative mRNA expression in EFNA4-Fc-treated EPHA10 shRNA expressing cells (n = 3) was normalized to that of control IgG-treated cells (n = 3). Bars represent SE; *p < 0.05; **p < 0.01; ***p < 0.001.