Fig. 3: MST3 suppresses YAP signalling by promoting STK25-mediated LATS1/2 phosphorylation and YAP ubiquitination. | Nature Communications

Fig. 3: MST3 suppresses YAP signalling by promoting STK25-mediated LATS1/2 phosphorylation and YAP ubiquitination.

From: ROS-induced cytosolic release of mitochondrial PGAM5 promotes colorectal cancer progression by interacting with MST3

Fig. 3

a Immunofluorescence for YAP in LOVO cells transfected with siLATS1/2 and/or flag-MST3 plasmids. Ratio of nuclear/cytoplasmic YAP was quantified. Vector+NC, n = 38 fields from 3 independent samples; Flag-MST3 + NC, n = 44 fields from 3 independent samples; Vector+siLATS1/2, n = 44 fields from 3 independent samples; Flag-MST3+siLATS1/2, n = 43 fields from 3 independent samples. Scale bar: 30 µm. P = 0.004. b qRT-PCR for YAP, CTGF (P = 0.04; P = 0.0041) and CYR61 (P = 0.03; P < 0.001) in LOVO cells transfected with siLATS1 and/or flag-MST3. n = 3 independent samples. c qRT-PCR for MST3 (P < 0.001) and LATS1 (P = 0.0053) in LOVO cells transfected with siLATS1 and/or flag-MST3. n = 3 independent samples. d Co-IP assay for endogenous interaction between MST3 and STK25, MST1, MOB1, YAP and LATS1/2 in HCT116 cells after immunoprecipitation with anti-MST3 antibody. e Western blotting for MST3, STK25 and pSTK25T190 in HCT116 cells transfected with flag-pcDNA3.1 or flag-MST3 plasmids. β-Actin was used as the loading control. f Western blotting for MST3, STK25 and pSTK25T190 in HCT116 cells transfected with pcDNA3.1-MST3T178E or pcDNA3.1-MST3K53A plasmids. β-Actin was used as the loading control. g Western blotting for MST3, STK25, pSTK25T190, YAP, pYAPS127, LATS1/2, pLATS1S909 and pLATS1T1079 in HCT116 cells transfected with siSTK25 and/or HA-MST3 plasmids. β-Actin was used as the loading control. h, i Western blotting for YAP in HCT116 cells transfected with NC/siMST3 (h) or pcDNA3.1/pcDNA3.1-MST3 (i), and treated with CHX (cycloheximide, 50 µg/mL) at indicated time points. β-Actin was used as the loading control. Relative gray value of YAP was calculated. j Co-IP analysis for binding between MST3 and 14-3-3. k Ubiquitination of YAP was measured 36 h after transfection with myc-Ub, flag-YAP and HA-MST3 in LOVO cells. l Western blotting for YAP, 14-3-3 and MST3 level in LOVO cells. Cells were transfected with different doses of HA-14-3-3 plasmid after treatment with NC or siMST3. β-Actin was used as the loading control. Western blotting images are representative of experiments that were repeated at least three times with similar results. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis in panels b and c was performed with one-way ANOVA followed by Tukey’s test, and the rest was performed with two-tailed unpaired Student’s t-test.

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