Fig 2: Statins inhibit YAP activation through mevalonate pathway. (A) Heatmap depicting expression of YAP‐related genes of each condition. (B) Gene set enrichment analysis of DEGs by each statin in YAP‐related genes up‐regulated by HCV infection. (C) Mechanism of YAP inhibition by statins through the mevalonate pathway. (D,E) HCV‐infected Huh7.5.1 cells were treated with 50 μM atorvastatin for 2 days and subjected to western blot and immunofluorescence (*p < 0.05 compared with control [Ctrl]). (F) HCV‐infected Huh7.5.1 cells were treated with 50 μM ATO for 2 days in the presence or absence of geranyl‐geranyl pyrophosphate (GGPP) (*p < 0.05, **p < 0.01, ***p < 0.001 compared with negative control [blue bar]; ##p < 0.01, ###p < 0.001 compared with only ATO‐treated condition [orange bar]). Data are presented as mean ± SEM of n = 3 independent experiments. AGFG2, ArfGAP with FG repeats 2; AJUBA, ajuba LIM protein; ANKRD1, ankyrin repeat domain 1; ASAP1, ArfGAP with SH3 domain, ankyrin repeat and PH domain 1; BICC1, BicC family RNA binding protein 1; BIRC5, baculoviral IAP repeat containing 5; CDC20, cell division cycle 20; CTGF, connective tissue growth factor; CDH1, cadherin 1; CDKN2C, cyclin dependent kinase inhibitor 2C; CENPF, centromere protein F; CNN3, calponin 3; CRIM1, cysteine rich transmembrane BMP regulator 1; DAAM1, dishevelled associated activator of morphogenesis 1; DAB2, DAB adaptor protein 2; DAPI, 4´,6‐diamidino‐2‐phenylindole; DDAH1, dimethylarginine dimethylaminohydrolase 1; DLC1, DLC1 Rho GTPase activating protein; DUSP1, dual specificity phosphatase 1; DUSP12, dual specificity phosphatase 12; DUT, deoxyuridine triphosphatase; DYNLL1, dynein light chain LC8‐type 1; ECT2, epithelial cell transforming 2; EMP2, epithelial membrane protein 2; ETV5, ETS variant transcription factor 5; FGF2, fibroblast growth factor 2; FLNA, filamin A; FSCN1, fascin actin‐bundling protein 1; FSTL1, follistatin like 1; FZR1, fizzy and cell division cycle 20 related 1; GADD45B, growth arrest and DNA damage inducible beta; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GAS6, growth arrest specific 6; GGH, gamma‐glutamyl hydrolase; GLI1, GLI family zinc finger 1; GLS, Glutaminase; GLS2, glutaminase 2; HEXB, hexosaminidase subunit beta; HMG‐CoA, 3‐hydroxy‐3‐methylglutaryl coenzyme A; HMMR, hyaluronan mediated motility receptor; INHA, inhibin subunit alpha; ITGB5, integrin subunit beta 5; LATS2, large tumor suppressor kinase 2; MAPK13, mitogen‐activated protein kinase 13; MARCKS, myristoylated alanine rich protein kinase C substrate; MDFIC, MyoD family inhibitor domain containing; mRNA, messenger RNA; MST1, macrophage stimulating 1; MYC, MYC proto‐oncogene, bHLH transcription factor; NDRG1, N‐myc downstream regulated 1; NES, normalized enrichment score; NRP1, neuropilin 1; PCNA, proliferating cell nuclear antigen; PDLIM2, PDZ and LIM domain 2; PHGDH, phosphoglycerate dehydrogenase; PMP22, peripheral myelin protein 22; PTCH1, patched 1; PXMP2, peroxisomal membrane protein 2; SGK1, serum/glucocorticoid regulated kinase 1; SH2D4A, SH2 domain containing 4A; SHCBP1, SHC binding and spindle associated 1; SHH, sonic hedgehog signaling molecule; SLIT2, slit guidance ligand 2; SMO, smoothened, frizzled class receptor; SMOX, spermine oxidase; STK4, serine/threonine kinase 4; STMN1, stathmin 1; TAZ, tafazzin; TGFB2, transforming growth factor beta 2; TGM2, transglutaminase 2; THBS1, thrombospondin 1; TK1, thymidine kinase 1; TNS1, tensin 1; TOP2A, DNA topoisomerase II alpha; TSPAN3, tetraspanin 3; WWTR1, WW domain containing transcription regulator 1; YY1AP1, YY1 associated protein 1; ZYX, Zyxin.

Fig 3: WDR1 promotes tumor cell proliferation and migration might through actin cytoskeleton-regulated YAP activity. A-D: The protein levels of total YAP and p-YAP were examined by western blotting analysis. Western blotting and quantification data showed that, Wdr1 depletion resulted in a significantly decreased level of total YAP, increased levels of pYAP (S127) and pYAP (S381) (A,B), whereas Wdr1 overexpression increased total YAP and decreased pYAP (S127), pYAP (S381) (C,D). E: Immunoblotting for the indicated proteins. Cell lysates were fractionated into nuclear and cytoplasmic fractions. TBP and GAPDH were shown as loading controls for each fractionation. shWDR1 cells exhibited dramatically decreased nuclear YAP and comparable level of cytoplasmic YAP. Quantitation of the relative protein levels was also shown. F: qPCR analyses of relative normalized expression of YAP target genes CTGF, ANKRD1 and CYR61. The expression levels of the three target genes were significantly decreased in shWDR1 A549 cells. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Activation of PAR2 promotes YAP stability. HEK293 cells were serum-starved for 12 h and then stimulated with (a) trypsin (1.5 nM or 2.5 nM) or (b) PAR2-activating peptide (AP) (100 μM) for different times. The level of total YAP, p127-YAP, and p397-YAP were measured with western blot. c HEK293 cells were serum-starved for 12 h and then stimulated with PAR2-AP (100 μM) or trypsin (2.5 nM) for 1 h. Expression of YAP mRNA was measured with real-time PCR. d Western blot was used to measure YAP protein expression in the nuclear and cytoplasmic fractions from HEK293 cell after PAR2 activation for different time. PARP (nuclear marker) and Hsp90 (cytoplasmic marker) were detected. e mRNA expression of YAP target genes were measured with real-time PCR. f Protein levels of YAP and PAR2 were examined with western blot in stable transfectant HT-29 cells with or without shRNA against PAR2 (sh-PAR2). g Expression of F2RL1 (PAR2), YAP, and (h) its target genes were examined with real-time PCR in stable transfectant HT-29 cells. i p127-YAP, p397-YAP, and total-YAP were measured with western blot in stable transfectant HT-29 cells. Note: To have equal amount of YAP protein, the lysates of HT-29-vector and HT-29-shPAR2 are loaded at the ratio of 1:2.5. Data were shown as mean ± SEM. N.S. no significance; *p < 0.05; **p < 0.01; ***p < 0.001 vs. corresponding control

Fig 5: GPRC5A promotes hypoxic cell survival via a novel HIF‐GPRC5A‐YAP axis AHypoxia‐induced YAP stabilisation via Ser397 dephosphorylation was abrogated in GPRC5A‐depleted cells.BHypoxia‐induced nuclear localisation of YAP was attenuated in GPRC5A‐depleted cells.CHypoxia stimulated TEAD activity (8× GTIIC‐luc reporter), but this effect was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n = 3).DHypoxia reduced LATS activity and expression, but this was prevented by GPRC5A depletion.EConstitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A‐depleted cells.FThe YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n = 3 independent experiments).GYAP knockdown was sufficient to induce caspase‐3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.HCrystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A‐depleted cells were rescued by expression of an siRNA‐resistant GPRC5A cDNA (GPRC5Asi1R), but this was prevented by co‐depletion of YAP (n = 3 independent experiments).IExpression of an siRNA‐resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia‐induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.JGPRC5A depletion attenuated hypoxia‐induced BCL‐XL expression.KConstitutively active YAP (S127A) expression induced BCL‐XL expression and prevented caspase‐3 activation/PARP cleavage by GPRC5A depletion in hypoxia.LGPRC5Asi1R expression restored BCL‐XL expression and prevented the appearance of cleaved caspase‐3 induced by GPRC5A depletion in hypoxia.Data information: Asterisks (*) indicate non‐specific band. Level adjustments were made to images in Adobe Photoshop post‐acquisition for clarity (equal changes applied to the entire image). Representative examples of n = 3 independent experiments are shown; data are presented as mean ± SEM. One‐way ANOVA with Tukey's multiple comparisons test was carried out in (F and H).Source data are available online for this figure.