Cell-surface exposure of MT1-MMP is mediated by KIF5B and KIF3A/KIF3B, but not by KIF1C. (A-F) Confocal micrographs of macrophages overexpressing MT1-MMP–mCherry (A-F) and wildtype macrophages (H-N), each treated with siRNA against (A,H) control sequence, (B,I) KIF5B, (C,J) KIF3A, (D,K) KIF3B, (E,L) KIF5B and KIF3B, (F,M) KIF1C, or (N) MT1-MMP. Cells were fixed, but not permeabilized, and stained with primary anti-mCherry (A-F) or anti–MT1-MMP (H-N) antibody, respectively, and secondary Cy5-conjugated antibody to label MT1-MMP–mCherry (A-F) or endogenous MT1-MMP (H-N) on the cell surface. White scale bar indicates 10 μm for all images. (G) Ratio of surface MT1-MMP–mCherry based on Cy5 fluorescence versus total cellular MT1-MMP–mCherry based on mCherry fluorescence. Fluorescence intensities for the control siRNA were set to 100%. *P < .05, **P < .01 compared with control. Bars indicate mean values ± SD. (O) Fluorescence intensities of surface-localized endogenous MT1-MMP, based on Cy5 fluorescence. Fluorescence intensities for the control siRNA were set to 100%. **P < .01 compared with control. For all values, 3 × 30 cells were evaluated. For specific values, see supplemental Table 1. Bars indicate mean values ± SD. (P) Flow cytometric analysis of endogenous MT1-MMP on the cell surface. Macrophages were treated with siRNA against control sequence, KIF5B, KIF3A, KIF3B, KIF5B and KIF3B, KIF1C, or MT1-MMP and stained for surface MT1-MMP and Alexa 488–labeled secondary antibody without fixation. Cells treated with control siRNA and stained with isotype IgG antibody were used as a negative control (gray). Note lower fluorescence intensity of cells transfected with siRNA against KIF5B, KIF3A, or KIF3B, compared with siRNA against KIF1C or unspecific control siRNA (green). Results from 1 of 2 respective experiments are shown. For specific values, see supplemental Table 1.