Fig 2: Azelnidipine can bind to MEK1 and MEK2(A) Computational docking model between Az and MEK1 and MEK2. (B–D) Az directly binds to MEK1 and MEK2. The recombinant proteins (B) or cell lysates of HEK293F (C), KYSE150, and KYSE450 cells (D) were incubated with Az-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. The results were analyzed by Western blot. (E) The binding capacity of Az to MEK1 and MEK2 in ESCC intact cells. The ESCC cells were treated with Az or DMSO for 24 h and treated with different temperatures. The protein bindings were visualized by western blot. (F) Cells ectopically expressing MEK1 (wild type [WT], mutant D147A, Q153A, or S194A) or MEK2 (WT, mutant D194A, F213A, or R238A) were incubated with Az-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. The results were analyzed by Western blot.

Fig 3: Azelnidipine inhibits the MEK-ERK signaling pathway(A and B) In vitro kinase assay of active MEK1 and MEK2 and inactive ERK1 and ERK2. The active MEK1 (A) and MEK2 (B), inactive ERK1 and ERK2, and ATP mixture were treated with Az or DMSO at 30°C for 30 min, and p-ERK1/2 T202/Y204 were visualized by Western blot. (C) The levels of MEK1/2-ERK1/2 signaling pathway in ESCC cells after Az (0, 1, 2.5, 5, and 10 µM) treatment. (D and E) Immunofluorescence staining of p-ERK1/2 T202/Y204 (D) and ERK1/2 (E) in ESCC cells after Az treatment. (F) Cell cycle was analyzed by PI staining, and the number of cells in each phase was analyzed by Modfit (n = 3). (G) The levels of Cyclin D1 and CDK6 in ESCC cells after Az (0, 1, 2.5, 5, and 10 µM) treatment. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: CUEDC2 inhibits STAT3-mediated osteogenesis by regulating SOCS3 protein stability.a–c The level of SOCS1, SOCS3, and phosphorylated STAT3 proteins was evaluated by western blotting. The relative protein levels of the indicated proteins were calculated after normalization to ß-ACTIN. *P < 0.05, **P < 0.01 versus control. a MC3T3-E1 cells were cultured for 6 days under the conditions shown in the Fig. 2b legend. b, c MC3T3-E1 cells transfected under the same conditions as the knockdown (Fig. 4a) of CUEDC2. After 24 h, cells were cultured with GM for 1 day. b The cytosolic and nuclear proteins were extracted from the cells for western blotting. MEK2 and LAMIN B were used as a loading control. The interaction between CUEDC2 and SOCS3 proteins (d) and the level of ubiquitin-conjugated SOCS3 proteins (e) were evaluated. HEK293T cells were co-transfected with indicated plasmids, as shown in d, e. Cell lysates were immunoprecipitated with anti-HA (d) or anti-SOCS3 or anti-UB (e) followed by western blotting with the indicated antibodies. As an input, 1% cell lysates were loaded. f, g Infection with Ad-shCUEDC2 or Ad-GFP was performed under the same conditions as described in the legend of Fig. 3b. After infection, the cells were cultured with Stattic (+, 2.5 µM; ++, 5 µM) in the presence of OM to investigate osteoblast marker gene expression (2 days), ALP activity (5 days), and mineralized nodule formation (12 days). **P < 0.01, ***P < 0.001 versus OM. ##P < 0.01, ###P < 0.001 versus Ad-shCUEDC2 with OM. f Real-time PCR analysis. g ALP and AR staining.

Fig 5: Tegaserod maleate inhibits the MEK1/2-ERK1/2 signaling pathway in GC. (A) MEK1 and MEK2 kinase activity was assessed by in vitro kinase assay using active MEK1, MEK2 and inactive ERK2 proteins. The effect of tegaserod maleate was determined using Western blotting. (B) The levels of p-ERK1/2, ERK1/2, p-RSK2 and T-RSK2 in HGC27 and AGS cells with different concentrations of tegaserod maleate (0, 0.25, 0.5, 1 and 2 µM) treatment for 24 h was determined by Western blotting. (C) The levels of p-ERK1/2 T202/Y204 were affected by MEK1 and MEK2 in HGC27 (C) and AGS (D) when treated with tegaserod maleate. ERK1/2 was immunoprecipitated by MEK1/2 and ERK1/2 was detected using p-ERK1/2 T202/Y204 (E) Immunofluorescence staining of HGC27 and AGS: cells were treated for 24 h, and then stained for p-ERK1/2 T202/Y204 (100 magnifications). The asterisks (**) (***) indicate a significant (p < 0.01 and 0.001).