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. 2023 Mar 14;13:1117326. doi: 10.3389/fonc.2023.1117326

Figure 2.

Figure 2

ADAM17 activity regulates EphA2 pS897 and total EphA2 levels through auto- and paracrine signaling. (A) ADAM17 inhibition with the mAb MEDI3622 (200nM) abrogates EphA2 pS897 while (B) TMI-005 (25µM) additionally reduces total EphA2 levels. (C) CM derived from untreated and TMI-005 (24h) treated NCI-H358 cells was transferred on naïve NCI-H358 cells to assess the paracrine effect on EphA2 S897 phosphorylation. To exclude the effect of remaining TMI-005 in the CM, cells were additionally pre-treated for 2h with TMI-005 as indicated in the upper treatment row. (D) Protein lysates of NCI-H358 tumor cells transduced with a doxycycline-inducible shRNA system directed against ADAM17 (A17) or control (NT) was used to assess the effect of ADAM17 and IR (24h and 48h post-IR) on EphA2 S897 phosphorylation and total EphA2 levels. (E) CM derived from irradiated and sham-irradiated NT/A17 cells was transferred on naïve cells for 2h to elucidate the paracrine effect of ADAM17 knockdown on EphA2 S897 phosphorylation. For all experiments, α-tubulin was used as the housekeeping control. These figures are representative of several independent experiments. (F) CM derived from NT/A17 NCI-H358 cells was used to assess the effect of ADAM17 for attracting migrating tumor cells. Bar graphs represent the average number of migrated cells counted in three randomly chosen fields/transwell of three independent biological replicates. **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. For all immunoblotting experiments, α-tubulin was used as the housekeeping control. These figures are representative of several independent experiments.