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. 2021 May 25;9:659428. doi: 10.3389/fcell.2021.659428

FIGURE 2.

FIGURE 2

Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser206 and Ser216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser37, Ser45, or Ser47, because a SDC1 core protein is modified with CS chains according to our previous study (Nadanaka et al., 2018). Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells (n = 4, each) was examined by CytoTox-ONETM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells (n = 6) was investigated. Statistical significance was determined using Student’s t-test.