Figure 3.

Transition of plasminogen to active-form plasmin on the cell surface annexin A2/S100A11 complex. (A) Biotinylated plasminogen (bio-PLG) was evaluated for the dose used in its transition reaction to active-form plasmin in MDA-MB-231 cell culture. A treatment duration of 60 min was scheduled. To confirm the ripe plasmin band originating from the added bio-PLG, its reaction stopper tranexamic acid (TXA) (1 mg/mL) was used. (B) The temporal evaluation of the added bio-PLG was performed according to a protocol similar to that described in the former experiment (A), except for using only 10 µg/mL. (C) The effect of calcium (Ca²⁺, 10 mM) on plasmin and other protease activities was studied. The samples prepared from the cell membrane fractions under non-reducing [dithiothreitol (DTT) (−)] conditions were subjected to gelatin zymography. After running, the gels were soaked in a refolding buffer and then incubated with a reaction buffer with (lower) or without Ca²⁺ (upper). (D) Non-triple-negative breast cancer (TNBC) MCF-7 cells and the indicated TNBC cell lines were all treated with bio-PLG at a final concentration of 10 µg/mL for 60 min. The bio-PLG attached to the cell membrane and the manifestation of its active derivative in the individual cell membranes were detected by Western blotting (WB) and zymography. (E) HEK293T cells were co-transfected with HA-tagged full-length or any of several S100A11 variants lacking the C-terminal end (see Supplementary Figure 2C ) and Myc-tagged annexin A2. The transfected cells were further treated with bio-PLG (10 µg/mL, 60 min). After fractionation of the membrane compartment from each cell treated, half of the extracts were used for input detection and zymography experiments. The remaining half was subjected to a pull-down procedure using streptavidin-conjugated beads. After precipitation of the samples with the beads, bound proteins were analyzed by WB using streptavidin–horseradish peroxidase (HRP), HA antibody, or Myc antibody. (F) The indicated cells were all treated with bio-PLG (10 µg/mL, 60 min). TGF-β (10 ng/mL, 24 h) stimulation induced intrinsic lysyl oxidase-like 4 (LOXL4) production in the parental MDA-MB-231 cells. After fractionation of the membrane compartment from each cell treated, similar experiments were performed as in panel (E) except for the use of the indicated antibodies to evaluate the endogenous proteins of interest. Cont: non-engineered MDA-MB-231 cells. (G) In vitro, a cell-free reaction was performed to evaluate the effect of annexin A2/S100A11 complex on the emergence of plasmin from inactive plasminogen. The prepared, purified proteins were mixed and incubated according to the formula. After a 30-min reaction at room temperature, the samples were subjected to gelatin zymography.