Fig 2: Effect of YAP knockdown on various genes in carcinoma cell lines. (A) qPCR data showed that YAP depletion resulted in a reduction of WWTR1 (in H157 only), two YAP target genes, five cell cycle progression regulator and survival genes but a marked increase in DSG3 with a lesser effect on CDH1 and CTNNA1 that encode the AJ proteins E-cadherin and a-Catenin, in both cell lines (n = 12, pooled from three independent experiments with technical quadruplicate, error bar: SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 determined by one-way ANOVA). (B) Western blotting analysis for the indicated proteins in both lines treated with scrambled control and YAP-specific siRNAs alongside both parental cell lines (representative of two independent biological samples). GAPDH was used as a loading control. Note that YAP depletion caused a compensation of TAZ in H157 but not in the H413 line and showed increased expression in DSG3 gene and protein as well as other DSM proteins with little effect on E-cadherin and a-Catenin.

Fig 3: Enhanced cytoskeletal anchoring of the cadherin adhesion complex by cortical tension limits contact expansion in doublets. (A) Contact stress (sc) for control progenitor cell doublets and doublets exposed to 50 nM LPA after 10-min contact time. (Ctrl) N = 13 and total n = 17; (LPA50) N = 7 and total n = 12. ****P value = 3.6e-17 Student’s t test. (B) Separation force (Fs) for control doublets and doublets exposed to 300 nM Latr or 100 nM Jasp. Upper shows actin-rich tethers formed between the cells during separation in the presence of Jasp. (Ctrl) N = 3 and n = 10; (Latr) N = 1 and n = 8; (Jasp) N = 3 and n = 13. (Ctrl–Jasp) Not significant (t test with Bonferroni correction for multiple comparisons). ***(Latr–Jasp) P value = 6.96e-4; ****(Ctrl–Latr) P value = 1.92e-5. (Scale bar: 5 µm.) (C) Rim to center mean intensity ratios for F-actin, myosin II, and Ctnna1 as a function of contact time (1, 2, 5, and 10 min) in the presence or absence of Jasp. F-actin was visualized by Phalloidin with N = 3; (Ctrl) n = 5, 3, 3, and 3 (corresponding to the different contact times mentioned above); and (Jasp) n = 3, 4, 3, and 4. Ctnna1 was visualized by immunohistochemistry with N = 1; (Ctrl) n = 5, 3, 5, and 5; and (Jasp) n = 3, 3, 3, and 4. Myosin II was visualized by Myl12.1-eGFP expression with N = 1; (Ctrl) n = 9, 8, 7, and 8; and (Jasp) n = 12, 9, 11, and 8. The shadowed areas denote SD. (D) Exemplary Airy Scan images of Ctnna1 subcellular localization at the contact edge of doublets exposed to Latr or Jasp. Quantifications below show cluster mean intensity (Im) and volumes (V) of the 50 largest clusters of each cell–cell contact. Arrows indicate distribution means. N = 1; (Latr) n = 7; and (Jasp) n = 5. (Scale bar: 5 µm.) (E) Cell–cell contact size (Ac) of control doublets and doublets exposed to Jasp or Latr as a function of contact time. Dotted lines connect contact formation (0 min) with the first time point when data were collected. The shadowed area denotes SD with (Ctrl) N and n as in Fig. 1C. (Jasp) N = 4, n = (3 min: 6, 6 min: 6, 9 min: 16, 12 min: 16, 15 min: 16, 18 min: 16); (Latr) N = 4, n = (3 min: 21, 6 min: 21, 9 min: 21, 12 min: 21, 15 min: 21, 18 min: 9). If not stated otherwise, N corresponds to the number of experiments, and n corresponds to the number of cell doublets.

Fig 4: Distinct characteristics of OSCC lines in a correlation between YAP abundance and cell junctional components. (A) Heat map for direct comparison of the genes in five categories between H157 and H413 cell lines. Note that, despite the relatively low level of YAP1, H413 displayed a phenotype of active YAP signature with concomitant reduction of cell junctional genes except for CTNNA1 and NF2. In contrast, a phenotype with an inverse trait was shown in H157 that lacked the YAP signature albeit YAP1 expression was abundant. Besides, this line also presented elevated expression in many desmosome (DSM) genes. (B) Confocal microscopy of YAP staining in two carcinoma lines with the quantitation of relative nuclear (Nuc) versus cytoplasmic (Cyto) distribution shown on the right (n = 5, a representative of two independent experiments). (C) YAP/TAZ luciferase activity indicated a 5-fold increase in H413 compared to H157 cells (pooled from three independent experiments). (D) Gene–gene interaction analysis identified the statistically significant genes which had a positive correlation with YAP1. The network map was generated based on the Pearson correlation coefficient. Genes shown in the green were P < 0.05 significantly correlated with YAP1 and in blue were P < 0.01 significantly correlated with YAP1. Genes on the left of YAP1 are potentially in the upstream Hippo pathway and those on the right are the known YAP1 target genes. (D-i) is the overall correlation network map based on the analysis of 46 genes in ten cell lines. (D-ii, iii) are the networks in each carcinoma cell line. Note that several cell junctional genes were shown to be associated with YAP1 only in the H413 line but not in H157. (E) Comparison of the qPCR data (normalised against the H157 control cells). Note that YAP1 knockdown caused increased expression in DSG3 in both lines (n = 8, two independent biological samples with technical duplicates) (Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 determined by Student’s t-test for A–D and for E determined by two-way ANOVA for multiple comparisons).

Fig 5: Cortical tension triggers Ctnna1/F-actin clustering at the contact of cell doublets. (A) Rim to center intensity ratios of core components of the cadherin adhesion complex in doublets. The schematic in Left shows the rim and center regions of the cell–cell contact where the fluorescence mean intensities were measured in control doublets (red line) and doublets exposed to LPA 50 nM (blue line). Doublets were fixed and analyzed for each time point separately (1-, 2-, 5-, and 10-min contact time). F-actin was visualized by Phalloidin with n = 3; (Ctrl) n = 5, 3, 3, and 3 (corresponding to the different contact times mentioned above); and (LPA) n = 6, 5, 4, and 5. Ctnna1 was visualized by immunohistochemistry with n = 3; (Ctrl) n = 5, 3, 5, and 5; and (LPA) n = 4, 3, 4, and 3. Myosin II was visualized by Myl12.1-eGFP expression with n = 1; (Ctrl) n = 9, 8, 7, and 8; and (LPA) n = 5, 5, 4, and 2. Shadowed areas denote SD. (B) Exemplary subdiffraction limited confocal images of Ctnna1 subcellular distribution at the cell–cell contact of control doublets (Left) and doublets exposed to 50 nM LPA (Right). Quantifications below show cluster mean intensity (Im) and volumes (V) of the 50 largest clusters of each cell–cell contact. Blue shadows in Right denote control conditions. (Ctrl) N = 1 and n = 3; (LPA) N = 1 and n = 5. (Scale bar: 5 µm.) (C) CLEM images with F-actin visualized by phalloidin–Alexa-488 (green). Right shows zoomed-in images of the boxed region in Left. (Scale bars: 1 µm.) (D, Left) Electron microscopy (EM) images of electron-dense clusters (outlined with green) at cell–cell contacts in control doublets (Upper) and doublets exposed to 50 nM LPA (Lower). (Scale bar: 200 nm.) D, Right is a zoomed-in image of cadherin-like clusters with individual clusters depicted by light blue arrowheads. (Scale bar: 20 nm.) (E) Representative Airy Scan images of F-actin (red) and Ctnna1 (green) colocalizing in clusters at the cell–cell contact of control doublets (Upper) and doublets exposed to 50 nM LPA (Lower). Cell doublets were fixed after 30-min contact time and visualized by Phalloidin (F-actin) and immunohistochemistry (Ctnna1). If not stated otherwise, N corresponds to the number of experiments, and n corresponds to the number of cell doublets. (Scale bar: 2 µm.)