Fig. 4. Chronic TNFα exposure induces malignant growth and CSC phenotype in OKF6/tert cells expressing HPV16 E6. HPV16 E6 and E7 expression was transduced into hTERT-immortalized OKF6/tert cells by infection with retroviral vectors or empty vector (CTL) as a control. CTL: OKF6/tert cells transfected with empty vector, E6: OKF6/tert cells expressing E6, E7: OKF6/tert cells expressing E6.

a Ectopic expression of HPV16 E6 and E7 was confirmed by qPCR. b Expression of p53 in OKF6/tert cells expressing E6 and E7 was determined by Western blot analysis. Actin was used as a loading control. c CTL, E6 and E7 cells were exposed to TNFα (5 ng·mL⁻¹) for 4 months to generate CTL/TNF, E6/TNF and E7/TNF cells, respectively. Proliferation capacity in medium with physiological levels of Ca²⁺ (1.5 mmol·L⁻¹) was determined by cell counting. d Anchorage-independent growth ability was determined by soft agar assay. e Self-renewal capacity was determined by a tumor sphere formation assay. f Migration ability was determined by transwell migration assay. *P < 0.01 compared to E6 by two-tailed Student’s t test. g Levels of key stemness transcription factors, KLF4, Lin28, Nanog and Oct4, were measured by qPCR and normalized to the expression of GAPDH. *P < 0.01 and **P < 0.05.