Overexpression of SOX2 rescued the inhibitory effects on HNSCC cell behavior induced by AFF4 depletion. ( a ) Western blot analysis of SOX2 expression in SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown at 48 h post transfection. n = 3. ( b , c ) MTT assay for cell proliferation in SOX2-overexpressing SCC1 cells (b) and SCC23 cells (c) in response to AFF4 knockdown at 3, 5 days. n = 3 at each time point. ( d ) Flow cytometry assay for ALDH activity of SOX2-overexpressing SCC1 cells in response to AFF4 knockdown at 48 h post transfection. n = 3. ( e ) Sphere formation assay conducted to evaluate sphere formation capacity of SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown after 2 weeks of culture in suspension. Bar indicates 200 μm. n = 3. ( f ) Would healing assay for migration activity of SOX2-overexpressing SCC1 cells in response to AFF4 knockdown. n = 3. ( g , h ) Transwell assay for invasion activity SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown. n = 3. * P -value