Fig. 8

LPL oxidation limits spreading and filopodia formation. a Representative immunoblot showing siRNA-mediated knockdown of LPL in PC3 cells. Knockdown was assessed three days after siRNA transfection (n = 3). b Simultaneous knockdown and overexpression of LPL in PC3 cells. The knockdown of endogenous LPL and the overexpression of eGFP-fused LPL were analyzed by western blotting. GAPDH was used as a loading control (n ≥ 3). c Representative confocal images of control (eGFP), wt LPL eGFP-expressing and C101A LPL eGFP-expressing PC3 cells. Cells with simultaneous LPL knockdown and overexpression were seeded on poly-D-lysine-coated coverslips for 30 min to allow weak adherence. Then, the cells were treated with 25 µM H₂O₂ (lower panel) or were kept untreated (upper panel). Thereafter, the cells were stained for nuclei (DAPI, white), MYO10 (red), and F-actin (Sir-actin, blue), and confocal images were acquired (n ≥ 4). Scale bar = 10 µm; ×100 magnification. d Magnified image gallery showing LPL expression (eGFP) and MYO10 signals at the tips of filopodia (n ≥ 4). Scale bar = 5 µm; ×100 magnification. e The ROIs of single cells were automatically selected based on eGFP and SiR-actin signals. The calculated areas (ROIs)/cell were used as measures of cell spreading. At least 30 cells were imaged per sample in each of three independent experiments. f Quantification of the number of filopodia in untreated or H₂O₂-treated cells. To count filopodia, intensity profiles were drawn on the cell extensions. Filopodia were defined by an intensity profile with a two-fold greater mean intensity of MYO10 and SiR-actin than background. At least 25 cells/sample were imaged in four independent experiments. P-values were calculated by two-way ANOVA. The data are presented as the mean ± SEM (n ≥ 3; *p < 0.05, **p < 0.01, ***p < 0.001, ns nonsignificant)