Effect of ADAM10 inhibition or knockdown on CCL2-induced signaling, adhesion, and F-actin polymerization in THP-1 cells. (A-B) THP-1 cells were (A) treated with 10 µM GI254023X or DMSO for 15 minutes or (B) transduced with lentivirus encoding shRNA targeting ADAM10 or scramble shRNA. Subsequently, cells were assayed for CCL2-induced phosphorylation of ERK1/2 and p38 at the indicated time points by western blotting. Samples treated with DMSO and GI254023X were run on the same gel. Total ERK1/2 and p38 levels were determined in parallel (separate blots had to be used for pp38). Signals were quantified by densitometry, normalized to the expression of total kinase, and expressed in relation to the phosphorylation level of (A) the unstimulated control (0 minutes) for DMSO-treated THP-1 cells and (B) separately for each transduced THP-1 cell type. (C-F) THP-1 cells were pretreated with 10 µM GI254023X or 0.1% DMSO for 15 minutes and stimulated with CCL2 (3 nM) or left unstimulated. Subsequently, cells were assayed for (C) binding to coated fibronectin, (D-E) upregulation of α₅ integrin surface expression (D, representative histograms; E, quantification), and (F) Rho GTPase activation. Rho activation was quantified as active Rho protein in relation to total Rho protein as determined by western blotting. (G) THP-1 cells were treated with 10 µM GI254023X or transduced with lentivirus to downregulate ADAM10. DMSO and scramble shRNA were used as controls. Subsequently, cells were assayed for polymerization of F-actin by flow cytometry using fluorophore-labeled phalloidin. Results were expressed as percentage of the controls (DMSO or scramble). Quantitative data represent means ± SEM of 3 independent experiments. Crosses indicate significance among treated cells calculated using 1-way ANOVA and the Bonferroni post-test. Asterisks without a line indicate significant differences to the nontreated control analyzed by 1-sample t test.