Figure 2.

p18/LAMTOR1 is involved in YAP nuclear signaling. A, comparison of the YAP cytoplasmic/nuclear ratios (logarithmic scale) in preosteoblast cells that stably express mCherry-YAP^wt and scramble (sh-ctl) or shRNAs against p18/LAMTOR1 (sh-p18) grown on glass coverslips overnight. YAP subcellular localization was analyzed by confocal microscopy and quantified with the Fiji software. Data are the mean ± S.D. (error bars) of two independent experiments with n > 30 (two-tailed unpaired Student's t test). B, subcellular localization of mCherry-YAP^wt (red) in preosteoblast cells that stably express scramble (sh-ctl) or sh-RNAs against p18/LAMTOR1 (sh-p18). Scale bar, 10 μm. C, Western blot analysis of YAP, YAP^pS127, and p18/LAMTOR1 expression in the indicated preosteoblast cell lines. Band intensities were quantified with a Chemidoc CCD camera (Bio-Rad), and ratios were calculated with Image Laboratory software (Bio-Rad). Actin was used as an internal loading control. Results are representative of three independent experiments. resc, sh-p18 cells that express exogenous p18/LAMTOR1-GFP. D, comparison of YAP cytoplasmic to nuclear ratios (logarithmic scale) in preosteoblast cells (β1^f/f) that express p18-GFP or GFP-p18 after overnight growth on glass coverslips. YAP subcellular localization was analyzed by indirect immunofluorescence, and signal intensity was quantified from confocal images with the Fiji software. Data (mean ± S.D.) are representative of two independent experiments with n > 30 cells analyzed (two-tailed unpaired Student's t test). E, in vitro osteogenic differentiation of preosteoblast cells (β1^f/f) that stably express scramble (sh-ctl) or shRNAs against p18/LAMTOR1 (sh-p18). Alkaline phosphatase (ALP) and Alizarin Red S (ARS; to detect calcium deposition) staining were performed to monitor differentiation status at day (D) 0, 4, and 15, as indicated. ***, p < 0.0001.