Figure legends
Figure 1. Effect of knocking out MMP-2 gene on osteosarcoma cell migration . a. Left; fold change MMP-2 gene expression for U2OS WT and MMP-2 KO cells. Right; MMP-2 protein and activity levels for WT and MMP-2 KO cells, including excreted MMP-2 in cultured OPTI-MEM medium, validating our MMP-2 KO cells. b. Above; transwell migration assay of U2OS WT, MMP-2 KO, MMP-2 KO cells with exogenous MMP-2, and MMP-2 KO cells with WT cultured media containing secreted MMP-2. Below; quantification of number of cells migrated after 24 hours. c. Left; wound closure assays of U2OS WT and MMP-2 KO (n=3). Right; quantification of % wound closure at 24 and 48-hour time-points. Inactivating the MMP-2 gene significantly impeded the migration of U2OS cells, and the addition of exogenous or secreted active MMP-2 did not recover their migratory phenotype.
Figure 2. Role of MMP-2 in enhancement of cell migration by sublethal concentrations of doxorubicin. a. Left; percent cytotoxicity of doxorubicin at various concentrations (0, 0.2, 0.4, 0.6, and 1 µM). Right; percent cytotoxicity of doxorubicin at 48 hours, showing 0.4 µM doxorubicin as a sublethal concentration of doxorubicin for U2OS cells. b. Left; wound closure assays for U2OS WT -/+ 0.4 µM doxorubicin at 24 and 48-hours. Right; quantification of % wound closure at 24 (upper) and 48 (lower) hour time-points (n=3). c. Left; wound closure assays for U2OS MMP-2 KO -/+ 0.4 µM doxorubicin at 24 and 48 hours. Right; quantification of % wound closure at 24 (upper) and 48 (lower) hour time-points (n=3). Although the sublethal concentration of 0.4 µM doxorubicin enhances U2OS cell migration, this enhancement in cell migration is lost when MMP-2 gene is inactivated.
Figure 3. MMP-2 regulates Src phosphorylation at Tyr-416. a. Top; levels of Src phosphorylation at Tyr-416 and Tyr-527 after 0 and 0.4 µM treatment at 24 hours for U2OS WT. Bottom; quantification of pSrc at Tyr-416 (left) and pSrc at Tyr-527 (right) for U2OS WT.
b. Top; levels of Src phosphorylation at Tyr-416 and Tyr-527 after 0 and 0.4 µM treatment at 24 hours for U2OS MMP-2 KO. Bottom; quantification of pSrc at Tyr-416 (left) and pSrc at Tyr-527 (right) for U2OS MMP-2 KO (n=3). * p<0.05 in comparison to untreated WT control.
In the presence of a sublethal concentration of doxorubicin, Src activation and phosphorylation at Tyr-416 occurs in an MMP-2-dependent manner.
Figure 4. MMP-2 knockout upregulates expression of Src Family Kinase inhibitors. a. Fold gene expression of endogenous Src kinase inhibitors (CHK/MATK, Csk, CDC2), showing significant upregulation of CHK/MATK in U2OS MMP-2 KO, compared to WT cells (*p<0.05). b. Top; protein levels of CSK and CHK/MATK in U2OS WT and MMP-2 KO cells. Bottom; quantification of CSK and CHK/MATK protein levels to 𝛃-actin levels in WT and MMP-2 KO cells (n=3). c. Top; protein levels of CSK and CHK/MATK in 0.4 µM doxorubicin treated U2OS WT and MMP-2 KO cells. Bottom; quantification of CSK and CHK/MATK protein levels to 𝛃-actin levels in 0.4µM doxorubicin treated U2OS WT and MMP-2 KO cells. *p<0.05. Loss of MMP-2 gene caused significant re-expression of the endogenous SFK inhibitor, CHK/MATK.
Figure 5. Overexpression of CHK/MATK impedes osteosarcoma cell migration in the presence of a sublethal concentration of doxorubicin.a. Left; GFP-expression of U2OS WT + GFP-MATK stables, in comparison to U2OS WT, in both GFP and bright field (Scale bar = 200 µm). Middle; gene fold expression for CHK/MATK in WT+GFP-MATK compared to WT. Right; protein expression for CHK/MATK in WT and WT+GFP-MATK, validating expression in WT+MATK stables. b. Top; wound closure assays comparing untreated U2OS WT and U2OS WT+MATK stables, showing complete closure at 48 hours. Bottom; quantification of % of wound closure of WT and WT + MATK stables at 24 and 48 hours (n=3). c. Top; wound closure assays for 0.4 µM doxorubicin treated U2OS WT and U2OS WT+MATK stables. Bottom; quantification of % wound closure of U2OS WT and U2OS WT+MATK stables at 24 and 48-hour time points, showing significant inhibition of cell migration in the WT+MATK stables when treated with 0.4 µM doxorubicin.
Figure 6. Graphical abstract. Left; MMP-2 gene represses CHK/MATK expression. This suppression allows for activation of Src by sublethal concentrations of doxorubicin. Right; inactivation of MMP-2 gene allows for re-expression of CHK/MATK by its turn inhibit doxorubicin-induced Src activation and cell migration.