![Figure 1. Effect of metalloproteinase inhibitors and ADAM10/ADAM17 knockdown on THP-1 cell migration. (A-B) THP-1 cells were preincubated with varying concentrations of the inhibitors (A) GI254023X (GI) in or (B) TAPI-1 or appropriate dilutions of vehicle (dimethylsulfoxide [DMSO]) for 15 minutes and then assayed for cell migration in response to 3 nM CCL2. The number of migrated cells in the lower compartment was determined by measurement of endogenous glucuronidase activity, and the results are expressed in relation to cells receiving no chemoattractant and no inhibitor. Migration of cells receiving no chemoattractant is indicated by dotted lines. (C) THP-1 cells were pretreated with GI254023X (10 µM) or DMSO (0.1%) for 30 minutes. Subsequently, cells were either directly assayed for CCL2-induced cell migration or washed to remove free inhibitor. Washed cells were either directly investigated for CCL2-induced chemotaxis or further incubated for 60 minutes (recovery) before the assay. Results are expressed in relation to the untreated control receiving no chemoattractant (dotted line). (D) THP-1 cells were transduced with different lentivirus encoding scramble-shRNA (scr), ADAM10-shRNA (A10-650 and A10-1947), or ADAM17-shRNA (A17-2061 and A17-2646) and assayed for CCL2-induced cell migration. Results are expressed in relation to the scramble control receiving no chemoattractant (dotted line). (E) Following CCL2 stimulation in the presence of 10 µM GI254023X or 0.1% DMSO (15-minute pretreatment), cells were removed from the top of the chemotaxis membrane, and the remaining cells were stained in the chemotaxis membrane. (Left) Representative images and (right) quantitative data are shown. (F) Green fluorescent protein-expressing THP-1 cells were pretreated with 10 µM GI254023X or 0.1% DMSO and subsequently investigated for transmigration through dsRed-expressing ECV304 cells grown on transwell filters. THP-1 cells were (left) visualized by confocal microscopy and Z-stack analysis and (right) quantified within (pos.1) and below (pos.2) the ECV304 layer. Quantitative data represent means ± SEM of 3 independent experiments. Significance was calculated using 1-way ANOVA and the Bonferroni post-test and is indicated by crosses. Asterisks without a line indicate significant differences to the nontreated control analyzed by 1-sample t test.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/26/10.1182_blood-2013-09-511543/4/4077f1.jpeg?Expires=1724237542&Signature=BP7Y5aXBiIj3jvt4qqwHXSonSnQY-F~aFZUE4VLBaBLSenUcixc7K4o~Vg6OaUfO7dIRzPDtIWaGf21zClfg4o5F6AhMonNURk82XrivTNcW8wl68-iOQwR9DXj4T8or7qYbM4kpZPGxGCnGnL8TLwHaucSug6EZ4ygVXmnDZtlkVzffzxWDtJ7BaCO-tOQ3QM69z~G010FNkaPuB4fRq89POD6eGiaf5Ogi-AY1wdr3jRLJi8AhZmbSYd65FILFqagovrlaz7lQN7iSQJ-VNH~jk6G1t-99AygXdNaK7xVzW9Ibk5Q~r3lGnVKe2xV~kLFMdLLHK6XXJzmcCWfPDQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of metalloproteinase inhibitors and ADAM10/ADAM17 knockdown on THP-1 cell migration. (A-B) THP-1 cells were preincubated with varying concentrations of the inhibitors (A) GI254023X (GI) in or (B) TAPI-1 or appropriate dilutions of vehicle (dimethylsulfoxide [DMSO]) for 15 minutes and then assayed for cell migration in response to 3 nM CCL2. The number of migrated cells in the lower compartment was determined by measurement of endogenous glucuronidase activity, and the results are expressed in relation to cells receiving no chemoattractant and no inhibitor. Migration of cells receiving no chemoattractant is indicated by dotted lines. (C) THP-1 cells were pretreated with GI254023X (10 µM) or DMSO (0.1%) for 30 minutes. Subsequently, cells were either directly assayed for CCL2-induced cell migration or washed to remove free inhibitor. Washed cells were either directly investigated for CCL2-induced chemotaxis or further incubated for 60 minutes (recovery) before the assay. Results are expressed in relation to the untreated control receiving no chemoattractant (dotted line). (D) THP-1 cells were transduced with different lentivirus encoding scramble-shRNA (scr), ADAM10-shRNA (A10-650 and A10-1947), or ADAM17-shRNA (A17-2061 and A17-2646) and assayed for CCL2-induced cell migration. Results are expressed in relation to the scramble control receiving no chemoattractant (dotted line). (E) Following CCL2 stimulation in the presence of 10 µM GI254023X or 0.1% DMSO (15-minute pretreatment), cells were removed from the top of the chemotaxis membrane, and the remaining cells were stained in the chemotaxis membrane. (Left) Representative images and (right) quantitative data are shown. (F) Green fluorescent protein-expressing THP-1 cells were pretreated with 10 µM GI254023X or 0.1% DMSO and subsequently investigated for transmigration through dsRed-expressing ECV304 cells grown on transwell filters. THP-1 cells were (left) visualized by confocal microscopy and Z-stack analysis and (right) quantified within (pos.1) and below (pos.2) the ECV304 layer. Quantitative data represent means ± SEM of 3 independent experiments. Significance was calculated using 1-way ANOVA and the Bonferroni post-test and is indicated by crosses. Asterisks without a line indicate significant differences to the nontreated control analyzed by 1-sample t test.
