Characterization of Protrudin-KO cell lines with or without stable expression of GFP-Protrudin, antibody validation, and verification of siRNA-mediated protein depletion. (A) KO of Protrudin in MDA-MB-231 cells was performed by CRISPR/Cas9-mediated genome editing. Stable expression of GFP-Protrudin in two KO clones was generated by lentiviral transduction. The indicated cell lines were either seeded on coverslips for confocal microscopy or lysed and subjected for Western blotting to detect protein expression levels of Protrudin. Cells grown on coverslips were immunostained for LAMP1. For Western blotting, anti-Protrudin and anti-actin (loading control) were used. Note that LAMP1-positive LE/Lys cluster perinuclearly in Protrudin-KO cells as expected, but not in Protrudin/GFP-Protrudin positive cells, verifying the functionality of GFP-Protrudin. (B) MDA-MB-231 and MDA-MB-231-Protrudin-KO#2 cells were grown on coverslips coated with Oregon Green gelatin for 4 h, stained with anti-TKS5 and phalloidin/Alexa Fluor 647 (actin), and examined by confocal microscopy (Airyscan; same dataset as in Fig. 3 C). The number of TKS5-positive invadopodia per cell was quantified. Each plotted point represents one cell, and values represent mean ± SD. Parental, n = 19 cells; Protrudin KO#2, n = 15 cells from three independent experiments, unpaired two-sided t test. (C) Verification of MT1-MMP antibody specificity by confocal microscopy and Western blotting. MDA-MB-231 cells were transfected with siRNA targeting MT1-MMP or control siRNA for 3 d. Cells were either lysed and subjected for Western blotting or seeded on coverslips and stained with anti-MT1-MMP and Alexa Fluor 488/Phalloidin (actin). Note that the anti-MT1-MMP signal is virtually gone in knockdown cells. (D) Western blots verifying knockdown of Protrudin, TKS5, RAB7, or FYCO1 in MDA-MB-231 cells using lysates with the indicated siRNA-transfected cells and the respective siRNA control treatments. Actin or vinculin was used as a loading control.