Fig 2: Overexpression of the three factors, OSY, activated the Hippo-Yap pathway in OSY-iPS cells. (A) Results of qPCR experiments indicated that the mRNA expression levels of Mst, Lats1, Mob1, and Yap genes in OSY-iPS cells were significantly higher than those in HuAECs. **P<0.01, OSY-iPS vs. HuAECs; n=3. (B) Results of western blot experiments indicated that the expression levels of Mst, Lats1, and Mob1 in OSY-iPS cells were significantly higher than those in HuAECs. In addition, the phosphorylation modification of Mst protein (p-Mst) in OSY-iPS cells was also significantly higher than that in HuAECs.

Fig 3: STAT3 activation is involved in the crosstalk between Gas6/AXL and the Hippo‐YAP pathway. A, A correlation between STAT3 and Yes‐associated protein (YAP) mRNA expression in the HNSCC dataset from TCGA database was analyzed. B, P‐YAP was detected after treatment with 5 μmol/L cryptotanshinone, AKTi‐1/2, or SCH772984 (STAT3, AKT or ERK inhibitors, respectively) with 2% FBS for 24 h and 1000 ng/mL rhGas6 for 1 h. C, P‐STAT3 was detected after treatment with 2% FBS for 12 h and 1000 ng/mL rhGas6 for 1 h. D, P‐YAP was measured after si‐STAT3 transfection and 2% FBS treatment for 48 h and 1000 ng/mL rhGas6 for 1 h. E, YAP expression in nucleus and cytoplasm were analyzed after treatment with 5 μmol/L cryptotanshinone with 2% FBS for 24 h and 1000 ng/mL rhGas6 for 1 h. F, Relative mRNA expression of CCN2 and AREG were analyzed after si‐STAT3 transfection and 2% FBS for 48 h and 1000 ng/mL rhGas6 for 1 h. G, P‐LATS1 and p‐MOB1 were assessed after treatment with cryptotanshinone and rhGas6 as described above. H, Immunoprecipitation assay was performed using anti‐LATS1 antibody after treatment with 2% FBS for 12 h and 1000 ng/mL rhGas6 for 1 h. I, J, After 2% FBS and rhGas6 treatment, IP assay was performed using antibodies against p‐STAT3 and STAT3. K, IP assay was performed after STAT3WT or STAT3Δ705 plasmid transfection and 2% FBS treatment for 48 h and 1000 ng/mL rhGas6 for 1 h. L, YAP expression in nucleus and cytoplasm were analyzed after treatment with 2% FBS and 100 ng/mL rhIL‐6 for 24 h. M, A schematic diagram illustrating that Gas6/AXL‐induced STAT3 activation blocked the Hippo inhibitory regulation on YAP by competitive binding to LATS1, which induced YAP dephosphorylation, nuclear translocation, and transcriptional regulation. *P < .05, **P < .01, ***P < .001, according to Student t test

Fig 4: Empagliflozin regulates Hippo signalling in LX-2 cells. (a) Following different doses of EMPA treatment in LX-2 cells for 12 h, the protein levels of MST1, MST2, phospho-MST1/2, MOB1, phospho-MOB1, LATS1, and phospho-LATS1 were evaluated via Western blot. (b) Statistical analysis on the relative expression of the Hippo pathway in LX-2 cells. Data are expressed as means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Fig 5: C12orf59 induces YAP nuclear localization. (A) Western blotting was used to measure the phosphorylated levels of LATS1 (p-LATS1), MOB1 (p-MOB1) and YAP (p-YAP) in KYSE30 and KYSE410 cells with infection of control or C12orf59-expressing lentivirus. (B) Cytosolic and nuclear extracts from KYSE30 or KYSE410 cells with or without C12orf59 overexpression were subjected to western blotting for YAP. β-tubulin was used as the loading control for whole or cytosolic lysates, and LaminB1 was used as the loading control for nuclear extracts. (C, D) Expression of C12orf59 and the YAP target genes ANKRD1, CTGF, and CYR61 in control and C12orf59-overexpressed KYSE30 and KYSE410 cells were detected by real-time PCR. RPL19 was used as the internal control for real-time PCR. (E, F) Sub-cellular distribution of YAP (red) was examined in control and C12orf59-overexpressed KYSE30 and KYSE410 cells by immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 μm. BF, bright field. Data were presented as mean values with standard deviations, *p < 0.05; **p < 0.01; ***p < 0.001.