HGF induced a collagen I–dependent, functionally significant increase in MT1-MMP at the basal surface of polarized, epithelial cells. HGF induced a collagen I–dependent, functionally significant increase in MT1-MMP at the basal surface of polarized, epithelial cells. (A) Left: Representation of MT1F-pHluorin-RFP construct. Right: MDCK cells stably expressing MT1F-pHluorin-RFP were seeded in 3D collagen I (2 mg/ml) and cultured for 48 h without HGF (control). Culture was then continued for 20 h in the presence of HGF (50 ng/ml) (HGF). Cells were imaged with confocal microscopy for pHluorin (green) and RFP1 (red). Arrows point to MT1-MMP at the basal cell membrane. Representative images are shown. (B) MDCK cells were seeded on collagen I fibril, gelatin, or plastic. The cells were infected with Ad-MT1F at an MOI of five in the presence of GM6001 (10 μM). After 24 h, GM6001 was washed out and cells incubated a further 24 h in the presence or absence of HGF (50 ng/ml). Surface MT1F was stained in live cells on ice and imaged by confocal microscopy. MT1F is shown in green, actin in red. Confocal sections at the apical and basal surface are shown. Note that basal localization of MT1F can be observed upon HGF treatment when cells are cultured on collagen I. (C) Results from B were quantified by measuring the intensity of the 488-nm channel in identical volumes at the apical and basal surface in 10 confocal stacks per condition. Volumes were selected based on the signal in the channel representing actin. The percentage of total MT1F at the basal surface was calculated as described in the Materials and Methods section. Data are plotted as the mean ± SD. The tested conditions were compared with a Kruskal–Wallis rank sum test (P < 2 × 10−16) before localizing differences with a pairwise Wilcoxon test with Bonferroni–Holm's correction of the P-value to correct for multiple testing. The asterisks depict statistically significant differences (*P < 0.05). Col I: collagen I. Pia Pernille Søgaard et al. LSA 2019;2:e201800276 © 2019 Søgaard et al.