Fig 2: Active mutants of YAP, TAZ and MKL1 reverse the inhibitory effects of intranuclear actin on proliferation and migrationVSMCs were infected with Ad:Control, Ad:NLS-ActinR62D, Ad:YAP, Ad:TAZ or Ad:activeMKL1 as indicated. Total RNA was extracted 24 h later and analysed by RT-PCR for the indicated target genes (A). One day post infection, cells were labelled with Edu for 4 h (B) or scratch wounded and migration quantified by Incucyte real time migration assay (C–E).

Fig 3: YAP m5C modification depends on NSUN2.A YAP protein levels were measured by ELISA in LUAD and adjacent normal tissues. B, C YAP protein levels in different stage (B) and tumor diameter (C) LUAD tissues. D ROC curves of YAP for discriminating LUAD from non-tumor tissues. E The enrichment of m5C in A549, H1299, Wi-38, MRC-5 and BEAS-2B cells at indicated regions (P1: 1–700, P2: 701–1400, P3: 1401–2100, P4: 2101–2800 P5: 2801–3493) of YAP 3′ UTR was calculated as the percentage of the input RNA via RNA-IP using the anti-m5C antibodies. F The enrichment of indicated m5C writers in A549 and H1299 cells at P1 region of YAP 3′ UTR was calculated as the percentage of the input RNA via RNA-IP using the indicated antibodies. G YAP mRNA level was measured by qPCR in A549 and H1299 cells with indicated plasmids overexpressed. H YAP protein level was measured by IB in A549 cells with indicated plasmids overexpressed. The level of proteins was normalized to that of GAPDH, and the normalized level of proteins in negative control groups were arbitrarily set to 1. I NSUN2 protein levels were measured in LUAD and adjacent normal tissues. J Correlation between plasma exosome concentration and tissue YAP protein level in LUAD samples. K YAP m5C levels were measured in LUAD and adjacent normal tissues. L, M NSUN2 protein levels in different stage (L) and tumor diameter (M) LUAD tissues. N, O ROC curves of NSUN2 and YAP m5C for discriminating LUAD from non-tumor tissues. The data are shown as the means ± SD from three independent experiments. Images of IB are representative ones of three independent experiments. **p < 0.01 indicate statistical significance. The data from (A, C, I, K, M) were analyzed by a student’s t test. The data from (B, F, G, L) were analyzed by a one-way ANOVA test. The data from (E) were analyzed by a two-way ANOVA test. The data from (J) were analyzed by a Spearman rank-correlation analysis.

Fig 4: TPA separates YAP from its dependent transcription factors.(A) TPA (final concentration 16 μM) separated YAP from TEAD, CREB, p73 and Runx2, as measured by IF using the indicated antibodies in Bel-7402 cells. Scale bar, 20 μm. The bar graphs are shown as the percent of cells in each of three categories (nuclear > cytoplasm, nuclear = cytoplasm, and nuclear < cytoplasm) from 100 randomly counted cells. (B) TPA induced cytosolic expression of YAP while reducing the nuclear expression of YAP. Cytosolic and nuclear fractionation analysis was performed in Bel-7402 and Bel-7404 cells treated with DMSO or increasing concentrations of TPA (final concentration 16–32 μM). The representative WB images from three independent experiments are shown in the upper panel, and the relative ratio are shown in the lower panel. The data from the “DMSO” group are arbitrarily set to 100%. (C) TPA (final concentration 16 μM) inhibited the interaction between YAP and its dependent transcription factors. YAP was immuno-precipitated by anti-YAP antibodies, and co-immuno-precipitations of CREB, Runx2, TEAD, and p73 were measured by WB. The representative WB images from three independent experiments are shown in the upper panel. The relative ratios are shown in the lower panel. The data from the “DMSO” group are arbitrarily set to 100%. (D–F) TPA (final concentration 16–32 μM) dose-dependently dissociated exogenous YAP-FLAG from TEAD4-Myc (D), CREB-HA (E), or Runx2-HA (F). Exogenous YAP-FLAG was immuno-precipitated by anti-FLAG antibodies, and co-immuno-precipitations of TEAD4-Myc, CREB-HA and Runx2-HA were measured by WB using the indicated antibodies. The representative WB images from three independent experiments are shown in the upper panel. The relative ratios are shown in the lower panel. The data from the “DMSO” group are arbitrarily set to 100%. The data are shown as the mean ± SD from three independent experiments. *p < 0.05 and **p < 0.01, as analyzed using Student’s t test (Fig. 2A and C) and one-way ANOVA (Fig. 2B and D–F). Specifically in Fig. 2A, comparisons of the percent of the cells categorized into the “nuclear > cytoplasm” group between different treatments, as indicated, are also statistically analyzed.

Fig 5: Proposed mechanism of canthin-6-one on inhibition of RA-FLS migration and invasion. Cant limits synovial fibroblast migration and invasion and reduces joint damage by up-regulating the expression of Hippo pathway regulators, MST1 and LATS1, thus down-regulating the expression and activation of YAP (indicated by phosphorylation of YAP on Ser397 and Ser127) and TAZ, which results in reduced production of TNF-α-induced MMPs (including MMP1 and MMP3) and pro-inflammatory cytokines (IL-6 and IL-1β).