Fig. 6
SPI1 promotes mRNA hypertranscription in HNSCC through a time-dependent mechanism. Representative images (a) and quantification (b) of EU incorporation in CAL27 and CAL33 cells after stable knockdown of SPI1 using two independent shRNAs (n = 3 independent replicates). Red: EU-labeled nascent RNA; blue: DAPI-stained nuclei. Scale bar, 100 μm. c Relative per-cell mRNA yield in CAL27 and CAL33 cells, determined after mRNA purification and normalized by cell number (n = 3 independent replicates). Representative EU-stained images (d) and quantification (e) of SAS and FaDu cells with or without SPI1 overexpression (n = 3 independent replicates). f Relative per-cell mRNA yield in SPI1-overexpressing SAS and FaDu cells, based on poly(A)-selected RNA quantification (n = 3 independent replicates). g Time-course RT‒qPCR analysis of SPI1 expression in Dox-inducible Tet-ON SAS and FaDu cells following doxycycline treatment (n = 3 independent replicates). The data were normalized to that of GAPDH and expressed relative to 0 h. h Immunoblot analysis of the SPI1 and c-MYC protein levels. Representative images (i) and quantification of the fluorescence intensity (j) of cells with incorporated EU at the indicated time points after Dox induction (n = 3 independent replicates). The data are presented as the mean ± SD. Scale bars, 50 μm. In (a–c), the shNC group indicates cells transduced with a lentiviral vector carrying nontargeting shRNA. In (d–f), the NC group indicates cells transduced with an empty lentiviral vector as the control for SPI1 overexpression. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test or an unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
