Specific tetraspanins associating with MT1-MMP. (A) MCF-7-MT1 and MDA-MB-231 cells were lysed in 1% Brij 99 lysis buffer, and the indicated tetraspanins were immunoprecipitated (TSPAN, cocktail of tetraspanin antibodies against CD9, CD63, CD81, CD82, CD151, and TSPAN4). Eluates were analyzed by gelatin zymography and by Western blotting for MT1-MMP, using the anti-catalytic domain LEM2/15.8 antibody. Std, MMP-2 standard; pMMP-2, pro-MMP-2; aMMP-2, active MMP-2; n.s., nonspecific. All antibodies used in these and other immunoprecipitation experiments have been previously shown to immunoprecipitate their target antigens (Barnstable et al., 1978; Tachibana et al., 1997; Yang et al., 2002). (B) HT1080 cells stably expressing TSPAN12 and MDA-MB-231 cells were lysed in 1% Brij 99, cell surface proteins were immunoprecipitated, and eluates were analyzed by gelatin zymography and by MT1-MMP Western blotting using the anti-hinge AB815 antibody. cMT1, cleaved MT1. MT1 Standards are from MCF-7 cells (negative for MT1) or MCF-7 cells stably expressing MT1-MMP. (C) FLAG-MT1-MMP, MHC1, and CD147 were immunoprecipitated from MCF-7-MT1 cells (lysed in 1% Brij 99) and eluates were Western blotted for CD9 and CD81.

Tetraspanin effects on MT1-MMP subcellular localization. (A) MCF-7-MT1-GFP cells were treated with the indicated control (IgG and MHC I) and tetraspanin (CD9 and CD81) antibodies (50 μg/ml for 2 d), fixed (in 4% paraformaldehyde), blocked in 5% BSA/PBS, incubated at 20°C with Alexa Fluor 546-conjugated anti-CD9 antibody (ALB6), and analyzed by fluorescence or phase-contrast microscopy. Bar, 20 μm. White arrowheads indicate coclustering of MT1-MMP-GFP with CD9 or CD81. (B) MCF-7-MT1-GFP cells were transfected with control or CD9/CD81/TSPAN12 siRNAs and plated within fibrin gels 2 d after siRNA transfection. Fibrin gels were spiked with 1% Alexa 546-labeled fibrinogen before addition of thrombin to form the gel. After 2 d, cells were visualized by confocal microscopy. Green, MT1-MMP-GFP; red, fibrin matrix. Laser power was adjusted to show similar green intensities between control and tetraspanin knockdown cells. Bar, 20 μm. Horizontal (green) and vertical (red) lines within the central XY image correspond to the planes chosen for XZ (up) and YZ (right) images, respectively.

Quantitation of tetraspanin effects on MT1-MMP-dependent cell growth/invasion within 3D fibrin gels. (A) MCF-7-MT1 and MCF-7-VC cells were treated for 2 d with siRNAs, and then cells were embedded within fibrin gels for the indicated times. Representative images are shown. Bar, 200 μm. (B) The percentage of area of cell coverage was determined to yield mean ± SD (n = 3; *p < 0.05, **p < 0.005 compared with control at each time point). (C) MCF-7-MT1 cells were treated with siRNAs as indicated, and then 2D growth was assessed using an MTT proliferation assay. Results are mean ± SD (n = 3).

Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p < 0.01 compared with control). Efficient tetraspanin knockdown was obtained similarly to that shown in Supplemental Figure 3.

Tetraspanin effects on MT1-MMP–dependent pro-MMP-2 activation. (A) MCF-7-MT1 cells were treated with control or CD9/CD81/TSPAN12 (3xTSPAN) siRNAs and after 4 d, cells were plated and incubated overnight in serum-free media supplemented with conditioned media from MCF-7 cells stably expressing MMP-2. Eighteen hours later, conditioned media and lysates were collected and analyzed for pro-MMP-2 activation by gelatin zymography or actin expression by Western blotting (as loading control), respectively. Densitometry was performed on zymograms and results displayed as percentage of active MMP-2 ± SD (n = 3; *p < 0.01 compared with control). (B) HT1080 cells were transfected with control or CD81/CD151/TSPAN12 (3xTSPAN) siRNAs. After 4 d, cells were plated and then incubated overnight in serum-free media with or without 4 μM cyto D. Conditioned media were then collected and analyzed by gelatin zymography for pro-MMP-2 activation. Zymogram densitometry was performed (n = 4; *p < 0.05 compared with control). (C) MCF-7-VC cells were treated and analyzed as described in A (n = 2).

Tetraspanin effects on MT1-MMP expression. (A) TX114 cell lysates from MCF-7-MT1 samples in Figure 5A were blotted for MT1-MMP (AB815 antibody) and actin. Densitometry was performed on Western blots to yield indicated percent values relative to control lanes ± SD (n = 3; *p < 0.05 compared with control); cMT1 is cleaved MT1 lacking the catalytic domain (generated via an autocatalytic cleavage event). (B) MCF-7-MT1 cells were treated with control, CD81, or CD9/CD81/TSPAN12 (3xTSPAN) siRNAs. After 4 d, cell surface expression of MHC I, E-cadherin, α3 integrin, CD9, CD81, and MT1-MMP was determined by flow cytometry. For MT1-MMP, AB815 is anti-hinge antibody (detects both full-length and cleaved MT1-MMP) and LEM2/15.8 is anti-catalytic domain antibody (detects full-length MT1-MMP only). Quantitation of flow cytometry mean fluorescent intensity (MFI) values of MT1-MMP by using either AB815 or LEM2/15.8 antibodies is shown and expressed as percentage of control ± SD (n = 4; *p < 0.05 compared with control for each antibody). (C) Cell lysates from HT1080 samples in Figure 5B were blotted for MT1-MMP (AB815 antibody) or actin. Densitometry of total MT1-MMP was expressed as percentage relative to controls ± SD (n = 3; *p < 0.01 compared with control). cMT1 is cleaved MT1-MMP. MT1-MMP standard was prepared as described in Figure 1B. (D) HT1080 cells were transfected with control or CD81/CD151/TSPAN12 (3xTSPAN) siRNAs, and flow cytometry was performed 4 d after transfection for MHC I, CD98, CD81, CD151, and MT1-MMP (AB815 and LEM2/15.8) with cyto D stimulation 1 d before flow cytometry. Quantitation of flow cytometry MFI values of MT1-MMP is shown and expressed as percentage of control ± SD (n = 5; *p < 0.05 compared with control for each antibody).

Tetraspanins affect MT1-MMP trafficking and stability. (A) MCF-7-MT1 cells were transfected with control or CD9/CD81/TSPAN12 (3xTSPAN) siRNAs. After 4 d, the cell surface subset of MT1-MMP was analyzed by [³⁵S]Met/Cys pulse-chase (see Materials and Methods). Appearance and processing of cell surface MT1-MMP was quantitated by densitometry and plotted as raw densitometric values for both pro- and active MT1-MMP as line graphs. (B) Four days after siRNA treatment, total MT1-MMP was analyzed by [³⁵S]Met/Cys pulse-chase, and densitometry was performed on total labeled MT1-MMP protein and expressed as ratio compared with control at time zero ± SD (n = 3; *p < 0.05 compared with control at each time point). cMT1 is cleaved MT1. (C) MCF-7-MT1-GFP cells were transfected with control or CD9/CD81/TSPAN12 (3xTSPAN) siRNAs. Two days after siRNA treatment, cells were treated with either lactacystin (10 μM), MG132 (2.5 μM), pepstatin A (50 μM)/leupeptin (10 μM), bafilomycin A1 (25 nM), or chloroquine (50 μM) for 2 d. Cell lysates were then collected, and MT1-MMP was analyzed by Western blotting. Densitometry was performed on total MT1-MMP and represented as ratio of total MT1-MMP for 3xTSPAN/control for each inhibitor ± SD (n = 3; *p < 0.05 compared with no inhibitor). Note that all the lysosomal inhibitors stimulated MT1-MMP expression in control siRNA-treated cells compared with no inhibitor, consistent with normal degradation of MT1-MMP in lysosomes (data not shown). (D) Tetraspanin knockdown was confirmed by Western blotting of CD9 and CD81 from cell lysates. Decreased MT1-MMP expression with tetraspanin knockdown is shown by Western blotting. TSPAN12 mRNA was also appropriately decreased (data not shown).