SOCE blockade inhibits the membrane expression of MT1-MMP. (A) WM793 cells expressing control shRNA or STIM1 and Orai1 shRNA were plated onto a 6-well plate overnight. The cells were washed and incubated with starvation RPMI 1640 medium. The conditioned medium was collected at various time points as indicated. (left) MMP2 and MMP9 activities in the conditioned medium determined through gelatin zymography. (right) Quantitation of MMP2 zymography activity in control (ctrl) or STIM1- and Orai1 shRNA–expressing cells (STIM1 & Orai1 knockdown [KD]) at 6 h (mean ± SD from three independent repeats). Ctrl, control; RU, relative unit. (B) Effects of SOCE blockade with 100 µM 2-APB or STIM1 and Orai1 shRNA on plasma membrane expression of endogenous MT1-MMP. The plasma membrane MT1-MMP in control WM793 cells, 2-APB–treated cells, or STIM1 and Orai1 knockdown cells was labeled with sulfo-NHS-SS-biotin, affinity purified with NeutrAvidin, and detected by Western blotting. (C) Quantification of the results in B with densitometry (means ± SD from three measurements). (D) WM793 cells stably expressing MT1-MMP–EGFP were treated with or without 100 µM 2-APB for 2.5 h and stained with anti-p230 trans-Golgi antibody and DAPI to visualize trans-Golgi network and nuclei. White lines indicate cell boundaries. Bar, 20 µm. (E) Confocal micrographs showing that SOCE blockade with 2-APB or STIM1 and Orai1 knockdown resulted in entrapment of MT1-MMP in the Rab5-positive endosomes. WM793 cells stably expressing MT1-MMP–EGFP were transiently transfected with mRFP-Rab5 to visualize endosomal compartments. White and magenta arrowheads indicate membrane and endosomes, respectively. Bars: (main images) 10 µm; (magnified views) 2 µm. (F) Quantitation of the colocalization between MT1-MMP–EGFP and mRFP-Rab5 in WM793 cells using LAS AF lite V2.0 (means ± SD). Each cell was manually segmented using the region of interest selection tool. The background threshold was identical for all the images. The numbers of cells used for quantitation are indicated in the parenthesis. Representative results from three independent experiments are presented. ****, P < 0.0001. (G) Biotinylated MT1-MMP–EGFP was allowed to be endocytosed in the presence or absence of 100 µM 2-APB. The plasma membrane biotinylation was removed by MESNA treatment, and the endocytosed MT1-MMP–EGFP were purified with NeutrAvidin beads and detected by Western blotting. (H) MT1-MMP–EGFP recycling experiment showing the effects of SOCE blockade. Plasma membrane MT1-MMP–EGFP was labeled through biotinylation. Cells were lysed immediately (lane 2), lysed after an immediate MESNA treatment without endocytosis (lane 1), or allowed to undergo endocytosis for 30 min and then treated with MESNA (lane 3–7). Cells were then either lysed immediately (lane 3) or allowed to undergo recycling for 30 or 60 min in the presence or absence of 100 µM 2-APB as indicated and then treated again with MENSA (lanes 4–7). Biotinylated MT1-MMP–EGFP were purified with NeutrAvidin beads and detected by Western blotting.