Fig 2: MOB1 acetylation mediates oxidative stress regulation of the Hippo pathway. (A and B) H1299 cells were incubated in the culture medium with or without 1 mM NAC, and treated with 100 mg/mL CHX for indicated hours before harvesting. Cells were lysed and endogenous MOB1 were detected by western blot analysis with MOB1 Ab (A). The curves were plotted based on the gray value of the bands of endogenous MOB1 in (A) by software Image J and GraphPad Prism 6. Error bars represented means ± SD. According to two-tailed Student’s t-test,* is for P< 0.05 and ** is for P< 0.01. Images and statistical results were based on three independent experiments (B). (C and D) H1299 cells were transfected with FLAG-MOB1-K11R mutant plasmids, cultured in medium with or without 1 mM NAC and treated with 100 mg/ml CHX for indicated hours before harvesting. Cell lysates were subjected to western blot analysis with FLAG Ab and Actin Ab (C). The curves were plotted based on the grey value of the bands of FLAG-MOB1-K11R mutant in (C) by software ImageJ and GraphPad Prism 6. Error bars represented means ± SD. According to two-tailed Student’s t-test, ‘ns’ is for no significance, P> 0.05. Images and statistical results were based on three independent experiments (D). (E) FLAG-MOB1 and HA-Ubiquitin were coexpressed in H1299 cells and treated with 25 μM MG132 for 12 h combined with 1 mM sodium arsenite for 1 h, 1 mM H2O2 for 1 h or 1 mM NAC for 12–24 h before harvesting. Cell lysates were subjected to immunoprecipitation with M2-Beads (anti-FLAG), and then immunoblotted with the antibodies to Ubiquitin, FLAG and AcK11-MOB1. (F and G) Two groups of H1299 KO cells (each group consisted of six dishes) were transfected with FLAG-MOB1 (WT or K11R mutant) respectively, following by using different concentration of sodium arsenite to treat cells for 1 h before harvesting. Cells were lysed, implemented western blot analysis and analyzed by antibodies as indicated in the figure (F). The gray value of the bands of p-YAP (S127) and YAP was determined by software ImageJ. Relative amount of p-YAP (S127) / YAP was analyzed and plotted by GraphPad Prism 6. Error bars represented means ± SEM. According to two-tailed Student’s t-test,* is for P< 0.05, ** is for P< 0.01 and **** is for P< 0.0001. Images and statistical results were based on three independent experiments (G).

Fig 3: 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 4: Activation of YAP signaling by XMU-MP-1 promotes OE regeneration and functional recovery of the olfactory system after injury(A) Immunohistochemistry detected the expression of YAP in the OE of control- and XMU-MP-1-treated 2-month-old C57BL/6 mice at 0, 3, and 7 dpl.(B) Quantitative analysis of the relative YAP density as shown in (A) (n = 6 sections from 6 mice per group).(C) Western blot detected the expression of p-MOB1, MOB1, p-YAP, and YAP in OE of control- and XMU-MP-1-treated 2-month-old C57BL/6 mice at 7 dpl.(D and F) Quantitative analysis of the relative p-MOB1/MOB1 (D), p-YAP/YAP (E), and YAP (F) levels as shown in (C) (normalized with control group, n = 8 blots from 4 mice per group, Student’s t test).(G) H&E staining of the OE obtained from control- and XMU-MP-1-treated 2-month-old C57BL/6 mice at 0, 3, and 7 dpl.(H) Quantitative analysis of the thickness of OE as shown in (G) (n = 12 sections from 6 mice per group).(I) Quantitative analysis of the time to locate food of control-treated (n = 10 mice) and XMU-MP-1-treated 2-month-old C57BL/6 mice (n = 13 mice) in the buried pellet test at 7 dpl (Student’s t test).(J and K) Double immunostaining of Ki67 (green) and p63 (red) (J) or PH3 (green) and K5 (red) (K) in the OE of control- and XMU-MP-1-treated 2-month-old C57BL/6 mice at 0 and 3 dpl.(L and M) Quantitative analysis of the percentage of both Ki67+ and p63+ (L) or PH3+ and K5+ (M) cells over p63+ or K5+ HBCs as shown in (J) and (K) (n = 13 sections from 6 mice per group).(N) Immunostaining of GAP43 (green) in the OE of control- and XMU-MP-1-treated 2-month-old C57BL/6 mice at 7 dpl.(O) Quantitative analysis of the density of GAP43+ cells as shown in (N) (n = 12 sections from 6 mice per group, Student’s t test).Images of selected regions are shown at higher magnification. Data are mean ± SEM, two-way ANOVA with Bonferroni’s post tests unless otherwise indicated, compared with control group; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Scale bars, 20 μm.

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.