For MMP-9 and MMP-2 detection in the CM of NCI-H441 and NCI-H1993 shCont and shILEI cells upon HGF stimulus with or without crizotinib treatment cells were plated in 6-well plates. At 70–80% confluency, cells were crizotinib- (500 nM) or DMSO-treated for 30 min followed by medium change to FBS-free media with continued crizotinib supply and the addition of human HGF (40 ng/ml). 24-h CM were collected and concentrated as described for Western blot analysis and all samples were adjusted to the same protein concentration followed by equal volume gel loading.

For MMP-9 and MMP-2 detection in protein extracts, NCI-H441 and NCI-H1993 shCont and shILEI snap-frozen tumor pieces with or without crizotinib treatment were homogenized in lysis buffer (see Western blot analysis for composition), total protein concentration was determined and equal protein amounts were loaded.

A 7.5% acrylamide gel was prepared with 2 mg/ml final concentration of gelatin; samples were loaded in non-reducing sample buffer without boiling. After running, the gel was washed twice for 30 min each in washing buffer (2.5% Triton X-100, 50 mM TRIS-HCl, pH 7.5, 5 mM CaCl2, 1 μM ZnCl2), rinsed for 10 min in incubation buffer (1% Triton X-100, 50 mM TRIS-HCl, pH 7.5, 5 mM CaCl2, 1 μM ZnCl2) at 37 °C followed by incubation in fresh incubation buffer for 24–40 h at 37 °C. The gel was stained in staining solution (0.5% (w/v) Coomassie Blue, 10% acetic acid, 40% methanol) for 1 h, rinsed with water and incubated in destaining solution (10% acetic acid, 40% methanol) until white bands were clearly visible. 10 ng of recombinant MMP-9 (Thermofisher Scientific) was loaded as positive control. Gels were quantified using ImageJ.

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