Skip to main content
. 2021 Apr 8;23(4):415–428. doi: 10.1016/j.neo.2021.03.006

Fig. 2.

Fig. 2

Trop-2 cleavage by ADAM10. (A) Inhibition of ADAM10 activity by treatment of BxPC3 cells with chemical inhibitors. Western blot detections of ADAM10 (top) and Trop-2 (bottom) show accumulation of the mature ADAM10 and corresponding reduction of the 40-kD Trop-2 cleavage band only upon treatment with GI254023X (blue). Quantification of the 40-kD Trop-2 cleavage band upon treatment with protease inhibitors is shown on the right, as percentage of total Trop-2. Ponceau red staining, control of protein loading. (B) Reduction of Trop-2 cleavage upon treatment of MTE4-14/Trop-2 transfectants with the ADAM10 inhibitor GI254023X (blue). Quantification of the Trop-2 cleavage band was carried out, and the ratio between uncleaved (un, black) versus cleaved (cl, gray) Trop-2 is shown on the right panel. (C) ADAM10 mRNA levels (red) 48 h upon treatment with ADAM10 siRNA or control siRNA (human CD133) (black). Vector: vector-alone transfectants; A87-A88: mutagenized Trop-2 at the cleavage site. The 2−ΔΔCT algorithm was used to calculate the relative changes in gene expression. RNA levels of ADAM10 in Vector, wt Trop-2 and A87-A88 Trop-2 cells are shown here for cell group cross-comparison, and are intended as reference data for the functional assays shown in the next figures. (D) ADAM10 protein inhibition by shRNA (top). Corresponding reduction of the Trop-2 cleavage is shown (mid). Ponceau red staining, control of protein loading (bottom). (E) Western blotting of Trop-2 cleavage in MTE 4-14/Trop-2 transfectants. (left) Cells were seeded at the indicated fraction of full confluency, as normalized to 100%, or (right) cells were seeded at equal confluency and then lysed at different subsequent time points. Ponceau red staining, control of protein loading. MW markers are indicated. (F) Western blotting of MCF7 human breast cancer cells grown in adhesion or in suspension and lysed 5 d after seeding for analysis of the Trop-2 cleavage. Red arrow shows cleaved Trop-2. Ponceau red staining, control of protein loading. MW markers are indicated. (Color version of figure is available online.)