Fig. 1. Chronic TNFα exposure induces calcium resistance in HPV-immortalized oral keratinocytes.

a HPV16-immortalized HOK-16B and hTERT-immortalized OKF6/tert cells were exposed to TNFα (5 ng·mL⁻¹) in low-Ca²⁺ (0.15 mmol·L⁻¹) keratinocyte growth medium (KGM) for the indicated days, and the cell numbers were counted. b HOK-16B and OKF6/tert cells were exposed to TNFα (5 ng·mL⁻¹) for 4 months in low-Ca²⁺ medium to generate 16B/TNF and OKF/TNF cells, respectively. Then, the cell proliferation capacity in high-Ca²⁺ (1.5 mmol·L⁻¹) DMEM containing 10% serum was determined by cell counting. Cells were seeded at a density of 2 × 10⁴ cells and counted after the indicated incubation period. Passage-matched controls, HOK-16B and OKF6/tert cells, were used for comparison with 16B/TNF and OKF/TNF cells, respectively. c The effect of high Ca²⁺ on the expression of differentiation markers was determined by qPCR using HOK-16B and 16B/TNF cells. The cells were cultured in low- or high-Ca²⁺ medium for 2 days and harvested for the assay. *P < 0.01 compared to the low-Ca² group by two-tailed Student’s t test. d Effect of chronic TNFα exposure on the expression of HPV16 E6 and E7 was determined by qPCR using HOK-16B and 16B/TNF cells.