Figure 4. Calcium down-regulates the DNA damage response.

(a) Cell extracts were prepared from undifferentiated NHKC cells and cells that were differentiated with high calcium concentrations for 24, 48 and 72 hours. Calcium-induced differentiation was performed in the presence of dimethyl sulfoxide (DMSO) as a vehicle control or GSI (10 μΜ). Lysates were analyzed with the indicated antibodies. For the ATM analysis, the membrane was first analyzed with a pATM antibody and was then re-probed with an antibody against the total ATM protein (b–d) NHKCs were treated as in (a) and expression levels of the differentiation marker Involucrin and genes involved in the DDR pathways were determined by qRT-PCR. All graphs are representative of three independent experiments, and the values are expressed as the means ± SD of experiments conducted in triplicate. The significance of the differences was calculated using two-tailed Student’s t test, with the vehicle-treated sample as the reference, and one-way ANOVA by comparing the mean of each indicated column. In panel b, ****P < 0.0001 and **P = 0.002. In panel c, ****P < 0.0001. In panel d, ****P < 0.0001, **P = 0.001 and ***P = 0.0001.