Fig. 3: A subtype-specific negative correlation between partial EMT and PI3K signalling in vitro and in vivo.

A Heatmap of Spearman’s correlations between HNC cell lines (x-axis) and TCGA-HNC primary tumours (y-axis). The HNC lines were classified as atypical (FADU and CAL27), classical (A253), basal (SCC9 and SCC25) and mesenchymal (SCC15) based on subtype-specific TCGA-HNC gene signatures. The subtype annotation bar is shown on top of the heatmap and on the right for TCGA-HNC primary tumours. B PI3K/AKT/mTOR signalling-related mutations were identified using CCLE and OncoPrint. C Heatmap showing mRNA expression of EMT markers and PI3K/AKT/mTOR signalling in normal-OKF6, basal-SCC25 and mesenchymal-SCC15 cells. D RPPA analyses of PI3K/AKT/mTOR signalling and EMT markers. Expression levels are shown as fluorescence intensity values for differentially expressed proteins (one-way ANOVA p-value < 0.05) and highlighted in pink. Circles and colours represent the level of expression for each protein. E Schematic illustration of orthotopic xenografts of HNC cells in NSG mouse tongues. Cells transduced with a lentivirus-containing luciferase gene were injected into the tongue and mice monitored weekly using PerkinElmer IVIS® Spectrum imaging system. F Representative images of bioluminescence at 38 days post-orthotropic implantation. IHC analysis of p-EMT markers in SCC15 and SCC25 xenografts. The epithelial marker CDH1 was expressed in SCC25 while the p-EMT marker PDPN was detected in SCC15. Magnification, X40; scale bars, 50 μm. G WB analysis of YBX1 and phospho-YBX1 in the HNC lines. β-actin was used as loading control. Data are representative of n = 2 biological replicates. H Immunofluorescence (IF) of YBX1 and p-YBX1 in SCC15 and SCC25. DAPI was used as counterstaining. Magnification, X40; scale bars, 50 μm. I Cytoplasmic and nuclear localisation of YBX1 and pYBX1 were quantitated using ImageJ from three independent experiments. YBX1 was localised in the cytoplasm of both lines with low nuclear detection of pYBX1 in mesenchymal SCC15 and high pYBX1 in the nucleus of basal SCC25 cells. Data are shown as mean ± SEM. The comparison between the two groups was performed using an unpaired t test (**p-value = 0.0011, ****p-value < 0.0001). Source data are provided as a Source Data file. Schematic Fig. 3E was created using Biorender.com.