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

Fig. 3.

Fig. 3

Trop-2 proteolytic processing-defective mutant versus wild-type. (A) Western blotting analysis of KM12SM/Trop-2 transfectants was carried out using anti-Trop-2 antibodies against the cytoplasmic tail and extracellular domain, as indicated. The Trop-2 bands were recognized by both antiextracellular domain and anticytoplasmic tail antibodies, indicating that the Trop-2 signal belongs to transmembrane forms. Red arrow, cleaved Trop-2. Vector, vector-alone transfectants. Ponceau red staining, control of protein loading. MW markers are indicated. (B) Flow cytometry analysis of MTE 4-14/Trop-2 transfectants using the T16 anti-Trop-2 antibody. The A87-A88 Trop-2 mutant (red) and wt Trop-2 (blue) are expressed at comparable levels. Vector: vector-alone transfectants. (C) Trop-2 cleavage at the cell surface. Trop-2 cleavage was assessed upon cell-surface biotinylation on KM12SM metastatic colon cancer cells. Pull-down after cell-surface biotinylation was carried out to reveal the status of Trop-2 at the cell membrane. (left) total cell lysate, (right) cell membrane pulled-down material. Western blotting analysis was carried out with a rabbit polyclonal antibody directed against the extracellular domain of Trop-2, and shows that wt Trop-2 is completely cleaved. Quantitative cleavage reduction was observed for the A87-A88 proteolytic site mutant. Red arrow, cleaved Trop-2. Ponceau red staining, control of protein loading. MW markers are indicated. (D) Growth curves in vitro of KM12SM human colon cancer cells and MTE4-14 murine cells transfected with wt Trop-2 versus A87-A88 Trop-2 mutant. Bars, standard errors of the mean (SEM). (E) ADAM10 (left) and MMP9 (right) reduced RNA levels by siRNAs, as measured by real-time RT-PCR. (F) Cell growth curves of wt Trop-2, A87-A88 Trop-2 or vector-alone transfectants upon ADAM10 (red), MMP9 (blue) or control (black) siRNA-mediated inhibition. Bars, SEM. P value (ADAM10, left panel): ANOVA analysis. Stars indicate post-hoc Bonferroni's t test P values (***, P ≤ 0.001). (Color version of figure is available online.)