Fig 2: WWP1 promotes AMOTL2 phosphorylation by LATS.(A) 293T cells stably transduced with shGFP (control) or shWWP1 lentiviruses were transfected with the indicated DNAs, then lysates were analyzed by Western blotting. (B) 293T cells were transfected with the indicated DNAs, then lysates were analyzed by Western blotting. (C) 293T cells were transfected with the indicated DNAs, then lysates were analyzed by Western blotting. (D) Control or CRISPR/Cas9-mediated LATS1/2-knockout 293T cells were transfected with the indicated DNAs, then lysates were subjected to in vivo ubiquitination assay.

Fig 3: Systematic model of the Hippo/YAP signaling pathway affecting cytotoxic drug resistance in osteosarcoma cells. Methotrexate and doxorubicin increase MST1 degradation in osteosarcoma cells, decreasing LATS1/2 total protein level and YAP phosphorylation, resulting in enhanced nuclear translocation of YAP. Endonuclear YAP then promotes the transcription of its downstream targets involved in anti-apoptosis and proliferation, leading to elevated proliferation and resistance to methotrexate and doxorubicin

Fig 4: WWP1 promotes the phosphorylation of LATS1/2 without affecting their stability.(A) 293T were cells transfected with the indicated DNAs including SFB-LATS1, then proteins in the resulting lysates were pulled down using streptavidin beads followed by Western blot analysis to probe for phospho-LATS. (B) 293T were cells transfected with the indicated DNAs including SFB-LATS2, then proteins in the resulting lysates were pulled down using streptavidin beads followed by Western blot analysis to probe for phospho-LATS. (C) 293T cells transfected with GFP-WWP1 WT or C890A were subjected to cycloheximide (CHX) chase assay for the indicated times. The resulting extracts were analyzed by Western blotting. Immunoblot for TAZ, which has a relatively short half-life, was included to serve as a positive control for the experiment. (D) MCF10A cells stably transduced with FLAG-WWP1 WT or C890A were subjected to cycloheximide (CHX) chase assay for the indicated times. The resulting extracts were analyzed by Western blotting. Immunoblot for TAZ, which has a relatively short half-life, was included to serve as a positive control for the experiment.

Fig 5: Analysis of AMOTL2 phosphorylation.(A) 293T cells were transfected with FLAG-AMOTL2 and HA-LATS WT, ΔUBA, or kinase dead, and extracts were analyzed for the indicated proteins by Western blotting. (B) 293T cells were transfected with GFP-WWP1 and WT or S159A FLAG-AMOTL2, and extracts were analyzed for the indicated proteins by Western blotting. (C) Wild-type or CRISPR/Cas9–mediated LATS1/2 double-knockout 293T cells were transfected with HA-AMOTL2, and extracts were analyzed for the indicated proteins by Western blotting. (D) 293T cells were transfected with WT or S159A FLAG-AMOTL2 together with GFP-WWP1 and HA-Ub, and extracts were subjected to in vivo ubiquitination assays followed by Western blot analysis.