If a man will begin with certainties, he shall end in doubt; but if he will be content to begin with doubts, he shall end in certainties.

(Francis Bacon, 1561–1626)

Dear Editor,

In our recent article1 published by Cell Death and Disease, we identified DAX-1 as an androgen-target gene highlighting the existence of a functional androgen receptor/DAX-1/aromatase interplay in estrogen-dependent breast cancer cells. Our conclusion is based on multiple technical approaches. As reported in the article, DAX-1 expression in breast cancer cells in response to androgen treatment has been evaluated not only by western blotting and immunofluorescence assays, but also by mRNA analysis showing a strong increase of DAX-1 expression following androgen administration. Furthermore, the importance of androgens in DAX-1 regulation and function was additionally assessed by DAX-1 promoter studies (luciferase-reporter assays, site-directed mutagenesis studies, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay), and by studies on the effect of DAX-1 silencing or overexpression.

Anyway, we express our sincere appreciation to Lalli2 for taking the time to lay its concerns out regarding the use of the K-17 Santa Cruz Biotech antibody for DAX-1 visualization. In the very early step of our research study, in hormone-dependent MCF-7 breast cancer cell line, we compared the performance of several DAX-1 antibodies. Also in the study by Helguero et al.,3 only partially cited by Lalli2 in his letter, authors tested different DAX-1 antibodies, including K-17, by comparing antibodies specificity using the same protein preparations (Figure 1).3 In this paper, all of the antibodies tested detected DAX-1 in ovarian tissue lysate by western blotting, but none of them detected DAX-1 in DAX-1-negative HeLa or T47-D cells. Nevertheless, these two cell lines were stained with K-17 for immunofluorescence analysis. From the data of these experiments (Figures 1 and 2),3 the authors conclude that ‘.....in immunofluorescence studies, K-17 antibody might be cross-reacting with other nuclear epitopes’ and dismiss it in this type of analysis. However, the authors did not exclude the use of K-17 for western blotting analyses. In fact, K-17 was used in the study by Helguero et al.3 for further evaluation of DAX-1 in a DAX-1-positive cell line (HC11 mouse mammary epithelial cells) by western blotting analysis (Figure 4).3 In our experimental system, by using the K-17 antibody, we discriminated the specific DAX-1 band of ∼50 kDa MW, from the non-specific one at 60 kDa, as they did in Helguero et al.3 As shown in Supplementary Figure 1, specificity of the ∼50-kDa band was proved by DAX-1 silencing experiments. We also want to underline that choosing the K-17 antibody was further supported by the use of this antibody in several other studies.3, 4, 5, 6, 7, 8, 9 We agree with Lalli2 that in immunofluorescence analysis discrimination between specific and non-specific immunostaining signal is not possible although, in our study, an increase in DAX-1 nuclear signal upon androgen treatment can be seen. Anyway, taking into account this criticism, immunofluorescence studies, aimed to DAX-1 visualization, are reported along with western blotting analysis discriminating the ∼50-kDa DAX-1 band.

In conclusion, we would like to emphasize that our original aim was to demonstrate the existence of a novel androgen receptor-mediated mechanism controlling the expression of DAX-1 and consequently of aromatase in a hormone-dependent breast cancer cell line. To accomplish this, we have confidence that precise and consistent methodology has been applied. The title of the Letter to the Editor by Lalli2 is provocative but, based on the facts mentioned, of questionable scientific validity: does Lalli2 think that the study of DAX-1 function may just rely on its visualization?