Fig. 4

GD-induced D5P downregulation increases MYH9/LATS1 aggregation and LATS1 degradation to activate YAP. A, B Immunoblotting analysis of phosphorylated YAP (serine 127) and YAP in HCT116 cells cultured with normal medium, glucose-free medium or glucose-free medium supplemented with G (A) or D5P (B) for the indicated times. GAPDH or α-Tubulin was used as the loading control. C Immunoblotting analysis of LATS1 in the supernatants (Sup) and pellets of the HCT116 cell lysate treated with or without GD (0 mM, 4 h). GAPDH was used as a loading control. D Immunoblotting analysis of LATS1, YAP, LC3A/B and P21 expression in HCT116 cells under normal conditions or GD (0 mM, 4 h) with or without MG132 or 3MA. α-Tubulin was used as a loading control. E Guanosine supplementation diminished GD-induced LATS1 insolubilization. HCT116 cells were cultured with normal medium, glucose-free medium or glucose-free medium supplemented with G for 4 h. Tubulin was used as a loading control. F Coomassie blue staining and MS analysis of GD-induced LATS1-interacting proteins in HCT116 cells. The bands marked in the red boxes (left) were sent for MS analysis. G The effects of GD and G/GMP supplementation on the interaction between MYH9 and LATS1 in HCT116 cells were examined using Co-IP assay. H HCT116 cells were cultured with normal medium (CTRL), glucose-free medium or glucose-free medium supplemented with 5 mM G for indicated time before immunoblotting analysis of MYH9 and LATS1. G3BP1 was used as a loading control for the pellets. I Guanosine supplementation diminished KL-11743-induced LATS1 insolubilization. HCT116 cells were cultured under normal conditions, treated with or without KL-11743, or KL-11743 plus G supplementation for 4 h. PNP or actin was used as a loading control. J Immunoblotting analysis of the recruitment of LATS1 to actin in SW480 cells under normal conditions, GD (0 mM) or GD supplemented with G. K Representative fluorescence images of LATS1 aggregation and F-actin in LATS1-GFP overexpressing HCT116 cells under normal conditions, GD (0 mM) or GD supplemented with G for 3 h. DAPI served as the nuclear stain. L Representative immunofluorescence images and quantitative analysis of YAP nuclear localization in HCT116 NC, shMYH9 and shMYH10 cells treated with GD (0 mM) for 4 h. DAPI served as the nuclear stain. M Immunoblotting analysis of LATS1 in the supernatants (Sup) and pellets of HCT116 NC, shMYH9, and shMYH10 cells under normal conditions or GD (4 h). G3BP1 was used as a loading control. N Immunoblotting analysis of MYH9, LATS1, phosphorylated YAP (serine 127) and phosphorylated JNK in the supernatants (Sup) and pellets of HCT116 NC and shPGM2 cells treated under normal conditions, GD (4 h) or GD supplemented with G (4 h). GAPDH was used as a loading control. O Immunoblotting analysis of LATS1 and YAP in the supernatants (Sup) and pellets of HCT116 cells treated under normal conditions, GD (0 mM, 4 h) or GD supplemented with D5P. GAPDH was used as a loading control. P Schematic diagram depicting that D5P disrupts the interaction between LATS1 and MYH9/F-actin and recovers the inhibition of LATS1 on YAP. The experiments in (A–E), (G–J), and (M–O) were repeated twice independently. In (L), data are the mean ± S.D.; P values were calculated using a two-tailed unpaired Student’s t-test. ***, P < 0.001