Fig. 4: Loss of YBX1 inhibits cell proliferation in basal-like HNC cells with active PI3K signalling.

A CRISPR-Cas9 DOX-induced (1 μM, 72 hours) YBX1-knockdown in SCC25 resulted in ~80% downregulation (** two-sided p-value=0.0052) of YBX1 by Q-PCR (upper panel) and WB (lower panel). WB images were quantified using ImageJ (n = 2 independent experiments). B Compared to SCC25 ( + YBX1), SCC25 (–YBX1) grew fewer (* p-value=0.01) and smaller colonies (** p-value=0.0016) 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 quantified using ImageJ (n = 3 experiments). C Transwell invasion of SCC25 (–YBX1) cells (n = 3 experiments). The number of invading cells was quantified using ImageJ. Magnification, X40; scale bars, 500 μm, * p-value = 0.0144. D GO and KEGG term enrichments for significant DEGs showed enrichment for genes involved in G2/M checkpoint suppression and ribosomal biogenesis, activation of apoptosis and anti-tumour inflammatory responses. E Significant loss of E2F, YY1 and MYC/MAX target genes in SCC25 (–YBX1) using GSEA regulatory target gene sets. The adjusted significance was empirically determined by 1000 gene-set permutations. F RPPA analyses of PI3K/AKT/mTOR signalling and EMT markers in SCC25 (–/+YBX1) treated with EGF (100 ng/mL) for 30 mins. Expression levels are shown as fluorescence intensity values for differentially expressed proteins (one-way ANOVA p-value < 0.05) and highlighted in pink. G Bioluminescence imaging of SCC25 –YBX1 (pink) and SCC25 + YBX1 (black) tumours (n = 3 experiments, 3 mice/group/experiments) at 38 days post-implantation. The radiance of bioluminescence was measured weekly, and data shown as mean ± SEM at each timepoint (* p-value = 0.0179, ** p-value = 0.0018). Tumours were weighed at sacrifice, and data shown as mean ± SEM (** p-value = 0.0085). H Optical images showing significant growth inhibition of SCC25 (–YBX1) xenografts and H&E staining of undifferentiated SCC25 ( + YBX1) compared to SCC25 (–YBX1). IHC confirmed loss of YBX1 in SCC25 (–YBX1) tumours. Magnification, X40; scale bars, 50 μm. (A–C) and (G) 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.