Fig. 7.

Dkk1 mediates acid-induced senescence in esophageal epithelial cells. A: quantitative analysis of SA-β-Gal staining following chronic acid pulsing for 5 days in EPC2 cells (left). Between acid pulses, cells recovered in normal growth medium or in medium containing anti-Dkk1 Ab or human IgG. Values are means ± SE of 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01. Right: SA-β-Gal staining in EPC2 cells before and after treatment with rhDkk1. Arrowheads, SA-β-Gal-positive cells. B and C: SA-β-Gal staining and quantitative RT-PCR analysis of p16 mRNA levels in EPC2 cells subjected to acid pulsing as described in A. D: MTT assay following acid pulsing in EPC2 cells. No acid, cells not exposed to acid (control); acid, cells subjected to acid pulsing and allowed to recover in normal growth medium between acid pulses and during postacid period; acid + anti-Dkk1 Ab, cells subjected to acid pulsing and allowed to recover in growth medium containing anti-Dkk1 Ab between acid pulses and during postacid period; acid + human IgG, cells exposed to acid pulsing and allowed to recover in growth medium containing human IgG between acid pulses and during the postacid period. MTT assay was performed 0, 24, 48 and 72 h postacid. For 0 h, MTT reagent was added 1 h after seeding the cells. Values are means ± SD of 3 independent experiments performed in sextuplicate. *P ≤ 0.05. E: MTT assay of EPC2 cells following acid pulsing in the absence of Dkk1 blocking. Anti-Dkk1 Ab was added only in the postacid period.