TLNRD1 localizes to, and modulates, filopodia. (A and B) U2OS cells transiently expressing MYO10-mScarlet and GFP, GFP-TLNRD1, or GFP-TLNRD1-F250D were plated on fibronectin for 2 h, fixed, and imaged using a spinning disk confocal microscope. Representative images are displayed (A). Yellow square indicates region of interest, which is magnified on the right. Scale bars: (main) 25 µm, (inset) 5 µm. (B) The number of MYO10-positive filopodia per cell was then quantified (n > 125 cells, three biological repeats; ***, P = 0.002). (C and D) Efficiency of siRNA-mediated (oligos nos. 6 and 7) silencing of TLNRD1 in U2OS cells as detected using quantitative PCR (C) or Western blotting (D). To detect TLNRD1 protein levels by Western blot, TLNRD1 was first immuno-precipitated from cell lysate as indicated. (E) TLNRD1-silenced (oligos nos. 6 and 7) U2OS cells transiently expressing MYO10-GFP were plated on fibronectin for 2 h and fixed, and the number of MYO10-positive filopodia per cell was quantified (n > 150 cells, three biological repeats; ***, P < 0.001). (F and G) U2OS cells transiently expressing GFP, GFP-TLNRD1, or GFP-TLNRD1-F250D were plated on fibronectin for 20 min, fixed, and imaged using an Airyscan confocal microscope. Scale bars: (main) 10 µm, (inset) 5 µm. (G) The number of endogenous filopodia per cell was quantified (n > 108 cells, three biological repeats; ***, P < 0.001). (H and I) RAT-2 cells transiently expressing GFP, GFP-TLNRD1, or GFP-TLNRD1-F250D were plated on fibronectin for 2 h, fixed, and imaged using a spinning disk confocal microscope. Scale bars: (main) 20 µm, (inset) 5 µm. (I) The number of endogenous filopodia per cell was quantified (n > 129 cells, four biological repeats; ***, P < 0.001). P values were determined using a randomization test (see Materials and methods for details). siCTRL, siRNA used as control.