Fig. 5: γ-Actin and KIF18B regulate mTORC1 signaling through modulating lysosomal positioning.

A Immunofluorescence staining for LAMP1 (red) followed by counterstaining with DAPI (blue) and DiO (green) of NC, ACTG1 silencing, KIF18B silencing and simultaneous silencing cells. The merged images are also shown. Scale bar, 10 µm. Cells were categorized into perinuclear-dominant lysosomal distribution (>50% of LAMP1-positive signals localized in the perinuclear region, <5 µm from the nucleus) and peripheral-dominant distribution (>50% of LAMP1-positive signals localized in the peripheral region, >5 µm from the nucleus). Quantification is based on three independent experiments. B Immunofluorescence staining for mTOR (green) and LAMP1 (red) followed by counterstaining with DAPI (blue) of NC, ACTG1 silencing, KIF18B silencing, and simultaneous silencing cells. The merged images are also shown. Scale bar, 10 µm. C Analysis of p70 S6K amino acid sequence. p70 S6K contains 2 YXXθ motifs (labeled in red), which mediate sorting of proteins to lysosomes. D The half-life of p70 S6K in NC, ACTG1 silencing, KIF18B silencing and simultaneous silencing cells was detected by CHX chase assay. Relative p70 S6K protein level were graphed. E BEL-7402 cells were pretreated with CHX for 12 hours, and then the cells were treated with MG132 or chloroquine for the indicated times. Finally, expression of p70 S6K examined by western blot. F BEL-7402 cells were transfected with KIF18B siRNA, ACTG1 siRNA or both siRNA. 2 days post transfection, cells were subjected to western blot analysis of p70 S6K, mTOR, Raptor, and LAMP1 in lysosome fraction. Error bars indicate the mean ± SD of three independent experiments. P values were calculated by student t test, *P < 0.05, **P < 0.01, ***P < 0.001.