Fig. 3

NUAK1 inhibition induces peripheral lysosomal positioning. A, F Representative confocal images of lysosomes using an anti-Lamp1 antibody (lysosome marker) in MDA-MB-231 (A) and U87 cells (F) under HTH-01-015 treatment. MDA-MB-231 and U87 cells were serum-starved overnight followed by 90 min of pretreatment with DMSO or HTH-01-015 (10 µM) and non-stimulated or EGF-stimulated for 60 min. Cells borders were marked with a boundary. Red, Lamp1; Blue, nuclei. B, G Quantification of the distribution of Lamp1⁺-lysosomes from A and F, respectively. Each bar represents the mean ± SD, Student t test. C Representative z-stack projections of Lamp1⁺-lysosomes in MDA-MB-231 cells. D, H Representative confocal images of lysosomes in MDA-MB-231 (D) and U87 cells (H) under WZ4003 treatment. Cells were serum-starved overnight followed by 90 min of pretreatment with DMSO or WZ4003 (10 µM) before stimulation with EGF for 60 min. Green, Phalloidin (F-actin); Red, Lamp1; Blue, nuclei. E, I Quantification of the distribution of Lamp1⁺-lysosomes from D and H, respectively. Each bar represents the mean ± SD, Student t test. J Representative confocal images of lysosomes in MDA-MB-231 expressing HA-tagged NUAK1 and stimulated with EGF for 60 min. Cells borders were marked with a boundary. Green, HA-NUAK1; Red, Lamp1; Blue, nuclei. K IB of Cathepsin D under NUAK1 inhibition in U87 cells. L IB of Cathepsin D under NUAK1 depletion in MDA-MB-231 cells