Fig 2: Cell proliferation and migration are also modulated by Mst1 and Yap. a–c Proteins were isolated from MDAT-32 cells and then western blot was used to evaluate the expression of Cyclin D1 and CDK4. Adenovirus Mst1 (ad-Mst1) and shRNA against Yap (sh-Yap) were transfected into MDAT-32 cells. d, e EdU staining was used to quantify the number of proliferated cells in response to Mst1 overexpression and/or Yap knockdown. g, h Transwell assay was used to evaluate the number of migrated cells in response to Mst1 overexpression and/or Yap knockdown. i–g RNA was isolated from MDAT-32 cells and then qPCR was used to evaluate the transcription of Rac1 and ROCK1. Adenovirus Mst1 (ad-Mst1) and shRNA against Yap (sh-Yap) were transfected into MDAT-32 cells. *p < 0.05 vs. control group, #p < 0.05 vs. ad-Mst1 group and/or sh-Yap group

Fig 3: H2O2 induces the phosphorylation of MST1/2 and ablation of MST1 leads to the suppression of E-cadherin expression and scattered cell growth whereas ablation of MST2 inhibits integrin ß4 expression and facilitates cell migration. (a) MCF-10A cells were treated with an indicated dose of H2O2 or PL for 12 h. Cell lysates were subjected to Western blotting using a specific antibody as indicated and the quantitative data from two independent experiments were shown. **p < 0.01. (b–f) Stable MCF-10A cells expressing one of two different shRNAs specific for MST1 (shMST1-#1; shMST1-#2), MST2 (shMST2-#1; shMST2-#2) or a control shRNA (shGFP) were subjected to Western blotting using a specific antibody as indicated and the quantitative data from two independent experiments were shown (b). Stable cells were subjected to IHC for E-cadherin. Representative micrographs were shown. Scale bar = 50 µm (c). Cells were seeded in 6-well plates for 20 h, and cells were then fixed and stained using crystal violet (d, upper panel). Percentage of scattered cells was defined as the ratio of scattered cells (=3 cells per colony) to total cells counted from three independent experiments in duplicate. Scale bar = 100 µm (e); In parallel, cells were subjected to transwell assays for cell migration (d, lower panel). Scale bar = 100 µm. The quantitative data from migration assays were quantified and presented as the means ± S.E. from three independent experiments in duplicate (f); **p < 0.01; ***p < 0.001. (g) Single clones of MCF-10A cells with either MST1 or MST2 knockout were obtained by the Crispr-Cas9 system. MCF-10A-MST1KO or MCF-10A-MST2KO cells were subjected to Western blotting (left panel), a cell-scattering assay (middle panel) and transwell assays (right panel). Data were quantified and presented as the means ± S.E. from three independent experiments in duplicates. ***p < 0.001. (h) MCF-10A-MST1KO or MCF-10A-MST2KO cells were subjected to immunostaining using an antibody specific for E-cadherin (upper panel) or ITGB4 (lower panel). Nuclei were counterstained with DAPI. Scale bar = 50 µm. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: Reversible regulation of NAFLD with HCP/MST1 or HCP/siMST1 complexes. (a) Schematic diagram for injection models of HCP/MST1 or HCP/siMST1 or HCP/pDNA in NAFLD mice. C57BL/6 mice were fed a high-fat diet for 12 weeks before being injected with HCP/MST1 or HCP/siMST1 for 3 weeks. The tissues were harvested 6 weeks after NAFLD. (b) Phenotypic pattern of liver tissue captured by a digital camera. (c, d) Changes in body weight and liver weight/body weight in each group with various treatments. (e) Fasting blood glucose level. (f) Serum insulin levels. (g) Homeostasis model assessment-insulin resistance (HOMA-IR) level (h) ITT, insulin tolerance test. (i, j) Serum ALT and AST levels. (k–p) Lipid content in liver and serum (n = 3–6, *p < 0.05, #p < 0.01; control vs sham, HCP/MST1 vs HCP/pDNA, HCP/siMST1 vs HCP/pDNA. Sham: mice fed with a normal chow diet; pDNA, nonfunctional plasmid; MST1, MST1 protein-coding plasmid; siMST1, functional plasmid-encoding siRNA of MST1; ALT: alanine transaminase, AST: aspartate transaminase).

Fig 5: MTX and DOX decrease mammalian sterile 20-like kinase 1 (MST1) expression by altering its protein stability. a Western blotting representative image of mammalian sterile 20-like kinase 1 (MST1) total protein level in MTX- or DOX-treated osteosarcoma cells. MST1 protein level was decreased by MTX and DOX. b The MST1 mRNA levels in the control and MTX- or DOX-treated osteosarcoma cells were monitored via real-time polymerase chain reaction using specific primers. The error bars represent mean ± SD. Compared with control (t test, n = 3), **P < 0.01. MST1 mRNA did not decrease after MTX or DOX treatments. c U2OS cells were treated with 100 µg/mL of cycloheximide for the indicated periods. The endogenous MST1 protein levels were determined by western blotting (upper panels), and relative MST1 protein levels were normalized to those of GAPDH (lower panels). The error bars represent mean ± SD. Compared with control (paired t test, n = 3), **P < 0.01.MST1 degradation was increased by MTX and DOX in U2OS cells. CHX cycloheximide