Fig. 5: Effect of gene silencing of EHF in HNSCC cells.

A, B, and C After knocking out the indicated genes by CRISPR/Cas9 techniques, monoclones were established and examined by IB analysis (A), invasion assays (B), and chemoresistance assays in response to the indicated concentration (conc.) of docetaxel in OBC01 cells (C). D, E, and F OBC01 cells transfected with control siRNA (NC) or EHF siRNAs were subjected to IB analysis (D), RT-qPCR analysis (E), and chemoresistance assays in response to the indicated concentration (conc.) of docetaxel (F). The ratio of mRNA expression to GAPDH in cells transfected with control siRNA (NC) was indicated as “1” (E). Each value represents the mean ± s.d. of triplicate determinations from a representative experiment. Similar results were obtained in at least three independent experiments. p values were determined by Student’s t-test. *p < 0.01 (C and F). α-tubulin was used as a loading control (A and D). G and H Representative images of immunohistochemical staining with anti-EHF antibody are shown in normal tongue tissue (top left panel), tumor nest (top right panel), and the invasion front (bottom two panels). Arrowheads indicate basal layer cells in normal tissue. Black arrows, orange arrows, and blue arrows represent EHF-negative, EHF-weak, and EHF-positive cancer cells at the invasion front, respectively (G). After representative images at the invasion front were randomly selected, we evaluated EHF-positive or negative/weak cancer cells at the invasion front, which were further assessed according to the Anneroth’s criteria by pathologists (H). Student’s t-test was used to compare differences between groups. **p < 0.02.