Fig 2: The YAP/TEAD1 axis promoted anoikis resistance, migration, and invasion of GC cells(A) WB analysis revealed the inhibition of the YAP/TEAD1 pathway in MKN-45, 746T, and HGC-27 cells, as a consequence of GRP94 knockdown, while GRP94 overexpression activated the YAP/TEAD1 pathway in HGC-27 cells.(B) Verteporfin increased YAP phosphorylation, reducing TEAD1 expression, while GA-017 had the opposite effect.(C and D) Verteporfin (Ver) negated the anoikis resistance observed in HGC-27-GRP94 cells. Conversely, YAP activation increased the anoikis resistance in MKN-45-shGRP94 cells, as shown by FCM. The experiment was repeated three times.(E and F) Ethd-1/calcein blue AM staining was performed to assess the apoptosis rate of HGC-27 and MKN-45 cells, with the application of verteporfin and GA-017. The working concentrations of calcein blue AM and EthD-1 were 2 μM and 4 μM, respectively. Displayed images are at 20× magnification with a scale bar representing 250 μm.(G and H) A wound healing assay assessed the impact of YAP on GRP94 using verteporfin and GA-017. Scale bar: 250 μm at 20× magnification.(I and J) Verteporfin mitigated the enhanced migration and invasion capacities of HGC-27 cells induced by GRP94. In contrast, GA-017 offset the migration and invasion inhibitions in MKN-45 cells caused by GRP94 downregulation. The scale bar, 50 μm, 100× magnification. (ns, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Results are shown as mean ± SEM.).

Fig 3: Rescue experiments proved that hucMSCs treatment reduced mechanical stiffness by regulating circANKRD42–miR-136-5p–YAP1 signal pathway in vivo and in vitro. (A) AFM images depicted that BLM caused the roughness of the lung surface and the thickening of the lung tissues. hucMSCs treatment improved the lung tissue morphology, but this therapeutic effect was reversed by overexpressed circANKRD42. The effect of circANKRD42 mutation was weaker than that of overexpressed circANKRD42. (B) In the TGF-β1 group, the cells became slender and flat. Stress fibers arranged in parallel along the main axis of cells were observed in the group. In the hucMSCs-treated group, the cell shape significantly retracted and the corresponding fibers were reduced. Overexpressed circANKRD42 reversed the effect of hucMSCs treatment on cells. (C) Young’s modulus measurement showed that hucMSCs treatment decreased the force compared with that in the BLM group, but overexpressed circANKRD42 reversed the effect of hucMSCs treatment on the force. The effect of circANKRD42 mutation on the force was weaker than that of overexpressed circANKRD42. The measured average Young’s modulus was 1291.667 ± 82.735 MPa in the BLM group, 563.833 ± 31.822 MPa in the BLM+hucMSCs group, 520 ± 20.460 MPa in the BLM+hucMSCs+circANKRD42 BP group, 942.05 ± 28.203 MPa in the BLM+hucMSCs+circANKRD42 RP group, and 604.233 ± 29.259 MPa in BLM+hucMSCs+circANKRD42 Mut. (D) Young’s modulus measurement revealed that hucMSCs treatment decreased the force compared with that in the TGF-β1 group, but overexpressed circANKRD42 reversed the effect of hucMSCs treatment on the force. The measured average Young's modulus was 641.167 KPa in the TGF-β1 group, 389.467 KPa in the TGF-β1+hucMSCs group, 382.767 in the TGF-β1+hucMSCs+circANKRD42 BP group, and 495.433 KPa in TGF-β1+hucMSCs+circANKRD42 RP. (E) Young’s modulus measurement showed that hucMSCs treatment decreased the reaction forces of cells compared with that in the TGF-β1 group, but overexpressed circANKRD42 reversed the effect of hucMSCs treatment on the force. The measured average Young's modulus was 8.858 ± 0.526 KPa in the TGF-β1 group, 4.756 ± 0.306 KPa in the TGF-β1+hucMSCs group, 4.504 ± 0.160 KPa in the TGF-β1+hucMSCs+circANKRD42 BP group, and 6.092 ± 0.240 KPa in TGF-β1+hucMSCs+circANKRD42 RP. (F) Cytoskeleton staining with FITC-phalloidin depicted that cytoskeleton tension was weakened by hucMSCs treatment but enhanced by circANKRD42 overexpression. (G) hucMSCs treatment inhibited the expression levels of YAP1, Myo1c, and F-actin and increased p-YAP1 expression in vivo and in vitro. circANKRD42 overexpression increased the expression of YAP1, Myo1c, and F-actin and decreased p-YAP1 expression, thus reversing the effect of hucMSCs treatment in vivo and in vitro. The effect of circANKRD42 mutation was weaker than that of overexpressed circANKRD42. Each bar represents the mean ± SD; n = 6; *p < 0.05.

Fig 4: hucMSCs treatment weakened mechanical stiffness in pulmonary fibrosis. (A) AFM images showed that the surface of lung tissues was smooth in the sham group. BLM caused the lung surface to become rough and increased the lung tissue thickness. hucMSCs treatment improved lung tissue morphology. (B) AFM images displayed that the cells were spindle shaped with smooth surface in the normal group. After TGF-β1 stimulation, the cells became elongated and flat and their cell height/roughness increased. hucMSCs treatment improved the cell state. Stress fibers arranged in parallel along the main axis of cells were found in the TGF-β1 group. After hucMSCs intervention, these fibers were reduced. (C) Young’s modulus value increased in the BLM group compared with that in the sham group, and hucMSCs treatment decreased the Young’s modulus. The average Young’s modulus was 299.017 ± 21.021 MPa in the sham group, 1275.667 ± 89.502 MPa in the BLM group, 284.667 ± 18.778 MPa in the sham+hucMSCs group, and 471.183 ± 29.462 MPa in the BLM+hucMSCs group. (D) Young’s modulus increased in the TGF-β1 group compared with that in the normal group, and hucMSCs treatment decreased the Young’s modulus. The average Young's modulus was 254.583 KPa in the normal group, 697.217 KPa in the TGF-β1 group, 238.150 KPa in the normal+hucMSCs group, and 434.150 KPa in the TGF-β1+hucMSCs group. (E) Tests using colloid probe with ball stuck pressed cells were conducted to determine the reaction forces of cells based on Young’s modulus. The average Young’s modulus was 2.686 ± 0.349 KPa in the normal group, 8.742 ± 0.422 KPa in the TGF-β1 group, 2.660 ± 0.333 KPa in the normal+hucMSCs group, and 4.623 ± 0.349 KPa in the TGF-β1+hucMSCs group. (F) Cytoskeleton staining with FITC-phalloidin depicted that the cytoskeleton tension was aggravated by TGF-β1 treatment and alleviated by hucMSCs treatment. (G) hucMSCs treatment decreased YAP1, Myo1c, and F-actin expression and increased p-YAP1 expression in BLM-induced mice model and TGF-β1-activated cell model. Each bar represents the mean ± SD; n = 6; *p < 0.05.

Fig 5: Acetylcholine promotes localization of YAP and enhances the HIF-1α signaling pathway in pancreatic cancer cells exposed to hypoxia. (A, B) pshCont- or pshnAChR-α7-transfected MIA-PaCa-2 (A) and AsPC-1 (B) cells were treated with 5 μM acetylcholine or left untreated for 1 h and exposed to 20% or 0.5% O2. After a 24-h incubation period, cells were harvested and the indicated proteins in whole-cell lysates were analyzed by immunoblotting. (C, D) MIA-PaCa-2 (C) and AsPC-1 (D) cells were transfected with siCont or siYAP, incubated for 48 h, and exposed to 20% or 0.5% O2. After a 24-h incubation period, cells were harvested and the indicated proteins in whole-cell lysates were analyzed by immunoblotting. (E, F) MIA-PaCa-2 (E) and AsPC-1 (F) cells were transfected with siCont or siHIF-1α, incubated for 48 h, and exposed to 20% or 0.5% O2. After 24 h of incubation, cells were harvested, and the indicated proteins in whole-cell lysates were analyzed by immunoblotting. (G, H) MIA-PaCa-2 (G) and AsPC-1 (H) cells were treated with 5 μM acetylcholine or left untreated for 1 h, followed by exposure to 20% or 0.5% O2. After a 24-h incubation period, cells were harvested for preparation of nuclear and cytoplasmic protein extracts, and the indicated proteins were analyzed by immunoblotting. (I) Whole-cell extracts of MIA-PaCa-2 cells were immunoprecipitated with anti-HIF-1α or anti-IgG (negative control), followed by immunoblot analysis with anti-HIF-1α and anti-YAP antibodies. (J, K) siCont- or siYAP-transfected MIA-PaCa-2 (J) and AsPC-1 (K) cells were co-transfected with p3×HRE-luc and pRL-luc, followed by treatment with or without 5 μM acetylcholine for 1 h, and exposure to 20% or 0.5% O2 for 24 h. Luciferase activity measured in whole-cell lysates was normalized to that of Renilla luciferase. Data are presented as means ± SEM (****P < 0.0001; ANOVA). (L, M) siCont- or siYAP-transfected MIA-PaCa-2 (L) and AsPC-1 (M) cells were incubated with 5 μM acetylcholine or left untreated for 1 h and exposed to 20% or 0.5% O2 for 24 h. The HIF-1α target genes, PGK1, PDK1, LDHA, CA9 and SLC2A, in harvested cells were examined by qPCR using 18S rRNA as an internal control. Data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA). Ns indicates no significance.