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

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Copyright © 2021 Nadanaka, Bai and Kitagawa.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector (n = 4) and SDC1(NTF)-3xFLAG (n = 3), or proliferation of BT-549 cells overexpressing the empty vector (n = 5), SDC1(FL)-3xFLAG (n = 5), and SDC1(CTF)-3xFLAG (n = 5) was measured.