Phospho-TAZ (Ser89) Antibody - #AF4315

Fig. 7: Gαi3 regulates PKA-Hippo-YAP signaling axis in pancreatic cancer cells. The primary priPC-1 cells stably expressing the Gαi3 shRNA (“sh-Gαi3-s1”), the CRISPR/Cas9-Gαi3-KO construct (“ko-Gαi3-s1”), the Gαi3-expressing lentiviral construct (“OE-Gαi3”), alongside their respective controls (“shC”, “Cas9-C” or “Vec”) were established, and the expression of listed proteins were tested (A–D). Primary priPC-1 cells stably expressing the Gαi3 shRNA (“sh-Gαi3-s1”), as well as the corresponding control (“shC”) underwent cytoplasmic/nuclear protein extraction, and listed proteins in the described cell fraction lysates were measured (E, F), cells were also subjected to immunofluorescence (IF) staining, with representative images presented (G). The priPC-1 cells expressingsh-Gαi3-s1 were treated with H-89 (a PKA inhibitor, 7 μM) or 0.1% DMSO as vehicle control (“Veh”), and expression of listed proteins was shown (H–L). After specified cultivation periods, cell proliferation and migration were examined, with results quantified (I, J). The priPC-1 cells with“sh-Gαi3-s1” were engineered to express either a constitutively active Akt1 mutant (caAkt1, S473D) or a control vector (“Vec”), expression of the described proteins was assessed by Western blotting (K). The designations “Cyt” and “Nuc” refer to cytoplasmic and nuclear proteins, respectively. *P 

Figure 8 FAT1 knockdown effects in lung cancer cells. (A) Cell cycle assay showed that FAT1 knockdown induced G0/G1 phase arrest. (B) Western blot results verifying the effect of knockdown FAT1 on the FAK–YAP/TAZ signaling pathway. (C) Immunohistochemical validation of the effect of FAT1 knockdown on the FAK-YAP/TAZ signaling pathway. (D, E) Pictures of dissected mouse tumors and knocking down FAT1 inhibits tumor growth in mice. **P < 0.01, ****P < 0.0001.

Fig. 3Knockdown of HSP110 inhibits hypoxia-induced autophagy and YAP/TAZ-TEAD4 activity in mice. Relative mRNA level (a) and protein level (b, c) of HSP110 pulmonary arteries in lung tissues in each group (N = 8). d Double immunofluorescence staining of α-SMA (green) and HSP110 (red) in pulmonary arteries (N = 8). White scale bars, 50 μm; Yellow scale bars, 25 μm. White arrows pointed to α-SMA and HSP110 double-positive cells. e Protein levels of LC3II, LC3I, Beclin1, ATG5, ATG7 and p62 in pulmonary arteries (N = 8). f–h Quantitative analysis of relative protein ratio of LC3-II/I and relative protein level of Beclin1, p62, ATG5 and ATG7 (N = 8). i Double immunofluorescence staining of α-SMA (green) and Beclin 1 (red) in pulmonary arteries (N = 8). Scale bars, 50 μm. j Protein levels of p-YAP, YAP, p-TAZ and TAZ in pulmonary arteries (N = 8). k Quantitative analysis of relative protein ratio of p-YAP/t-YAP and p-TAZ/t-TAZ (N = 8). l–n Nuclear protein levels of YAP, TAZ and TEAD4 and quantitative analysis of relative protein level of nuclear YAP, TAZ and TEAD4 (N = 8). o Double immunofluorescence staining of α-SMA (green) and YAP (red) in pulmonary arteries (N = 8). White scale bars, 50 μm; Yellow scale bars, 25 μm. White arrows pointed to α-SMA and YAP double-positive cells. Data are means ± SD from 8 mice per group. *p 

Fig. 5.| Influence of Sal-B on inflammatory response during YAP/TAZ/JNK signaling pathway in ECs. (A) Expression levels of YAP, TAZ, JNK, NF-κB and TNF-α were monitored by RT-PCR (n = 3). (B–C, F) Proteins (YAP, p-YAP, TAZ, p-TAZ, JNK, Nuclear NF-κB P65, Total P65 and TNF-α) in the pathway were detected by western blot (n = 3).

FIGURE 6 The effect of the Hippo pathway on macrophage polarization. To investigate whether EMs exosomes@miR-196a-5p affects macrophage polarization through the Hippo pathway, we treated macrophages with EMs exosomes@miR-196a-5p in combination with the Hippo pathway inhibitor XMU-MP-1. (A) RT-qPCR was used to detect macrophage polarization markers (iNOS and CD86 for M1 markers; CD206 and CD163 for M2 markers). (B) Western blot was used to detect the protein expression levels of macrophage polarization markers (iNOS and CD86 for M1 markers; CD206 and CD163 for M2 markers). (C) The proportion of CD86+ (M1) and CD206+ (M2) macrophages was detected using a flow cytometer. (D) Western blot was used to detect Hippo pathway-related proteins (MST1, p-YAP1, YAP1, p-TAZ, and TAZ). Data represent mean ± SD from three independent experiments. XMU-MP-1: Hippo inhibitor. *: P < 0.05, **: P < 0.01, compared with EMs exosomes@miR-196a-5p.