Fig. 5: Cytoplasmic YBX1 induces p-EMT in the absence of PI3K signalling.

A CRISPR-Cas9 DOX-induced (1 μM, 72 hours) YBX1-knockdown in SCC15 cells resulted in ~80% downregulation (****p-value < 0.0001) of YBX1 by Q-PCR (upper panel) and WB (lower panel). WB images were quantified using ImageJ (n = 2 independent experiments) B Compared to SCC15 ( + YBX1), SCC15 (–YBX1) cells grew more and bigger colonies (* p-value = 0.0448) on ultra-low attachment plates for seven days. Images of spheroids were captured using an EVOS cell imaging microscope. The number and average size of colonies were quantitated using the ImageJ (n = 3 experiments). C Transwell invasion of SCC15 (–YBX1) cells (n = 3 experiments). The number of invading cells was quantitated using ImageJ. Magnification, X40; scale bars, 500 μm, * p-value = 0.0321. D Significant enrichments for the protein kinase B signalling and the translation initiation in SCC15 (–YBX1) cells using GO gene sets. The significance was empirically determined by 1000 gene-set permutations. E RPPA analyses of PI3K/AKT/mTOR signalling and EMT markers in SCC15 (–/ + YBX1) treated with EGF (100 ng/mL) for 30 mins. Expression levels are shown as relative fluorescence intensity values for differentially expressed proteins (one-way ANOVA p-value<    0.05) and highlighted in pink. F Bioluminescence imaging of live animals injected with either SCC15 (–YBX1) or SCC15 ( + YBX1) (n = 3 experiments, 3 mice/group/experiments) at 38 days post-transplantation. The radiance of bioluminescence was measured weekly, and data shown as mean ± SEM at each timepoint (*p-value = 0.0247). Tumours were weighed at sacrifice, and data shown as mean ± SEM. G Acceleration of orthotopic tumour growth for SCC15 (–YBX1) confirmed by H&E staining of tumours and IHC showing loss of YBX1 in primary and metastatic tumours compared to SCC15 ( + YBX1). Magnification, X40; scale bars, 50μm. (A–C) and (F) data are shown as mean ± SEM. The comparison between the two groups was performed using an unpaired two-sided t test (*p-value < 0.05, **p-value < 0.01, ****p-value < 0.0001). Source data are provided as a Source Data file.