Extended Data Fig. 3 |. p53-dependent miRNA in OCSCC.

a, OCSCC RNA transfer to neurons via EVs. Representative confocal immunofluorescence image demonstrating PCI-13 cell-derived RNA labelled with SYTO RNASelect (green) in the perinuclear cytoplasm of a neuron (labelled with β3-tubulin, red). Images were captured 12 h after application of EVs derived from PCI-13 cells labelled with SYTO RNASelect; data replicated in two independent experiments. b, EVs derived from PCI-13 cells contained mainly small RNA species. Bioanalyzer results showing presence of RNA in EVs from PCI-13 cells. Representative band of EV RNA by Agilent RNA Pico Chips; data independently replicated in ten experiments. c, An unsupervised hierarchical clustering heat map showing differentially expressed EV miRNAs between p53-isogenic PCI-13 cells. p53^WT, n = 3 biologically independent samples; p53^null and p53^mut, n = 14 biologically independent samples. d, Heat map of differentially expressed miRNA, arranged by unsupervised hierarchical clustering, presenting the miRNA sequencing for EVs derived from isogenic PCI-13 cells expressing p53^WT versus no p53 (p53^null) or mutant p53 (p53^C238F, p53^G245D, and p53^R273H). The Pearson distance and Ward’s minimum variance method were used for pairwise clustering (c, d). Red and green indicate increased and decreased expression levels, respectively (n = 2 to 5 per group). e, Fold change in hsa-miR-141-5p and hsa-miR-34a-5p in EVs derived from p53^WT PCI-13 cells (blue, n = 3 biologically independent samples) compared with p53^null or p53^mut cells (red, n = 14 biologically independent samples). Results are log₂ normalized. f, Real-time PCR quantification of miR-34a and miR-141 in ventral tongues from Trp53^flox/flox and Krt5^CreTrp53^flox/flox mice (n = 7 per group). g, Real-time PCR quantification of CDK6 (miR-34a target) and ZEB1 (miR-141 target) in neurons treated with antagomiR-34 or antagomiR-141 compared with nonspecific antagomiR-treated controls (n = 3 biologically independent samples per group). h, Quantitative validation of miR-34a and miR-141 overexpression after transfection with miR-34a and miR-141 mimics, respectively. TG neurons were transfected with miR-34a mimic, miR-141 mimic, or scramble miR, and overexpression of miR-34a and miR-141 was confirmed by real-time PCR (n = 7 biologically independent samples per group). i, Real-time PCR quantification of miR-34a in orthotopic tumour xenografts of HN30 OCSCC cells treated with shControl (blue) or shmiR-34a (purple). n = 4 biologically independent samples per group. j, k, Western blot of NOTCH1 (confirmed miR-34a target) in OCSCC transfected with lentiviral miR-34a inhibitor or scramble miRNA inhibitor (j). Bar graph quantification of the blots demonstrates no impact of miR-34a inhibition on p53 expression and is normalized to the total amount of β-actin (n = 4 biologically independent samples per group, j). Unpaired two-tailed t-test; bars and dot plots represent mean ± s.e.m. (e–i, k).