Expression of NOSs in xenografts of HeLa cells.

<p>A, B. Expression levels of nNOS mRNA (A) and iNOS mRNA (B) in HeLaXs. *, **and *** indicate significant differences between experimental and control xenografts, and between HeLaXs and HeLa cells by ANOVA-test (A) and by Student's t-test (B), at *, P<0.05, **, P<0.01, ***, P<0.001, respectively. The letter a means significant differences between average levels of nNOS mRNA and iNOS mRNA in HeLa cells by Student's t-test, at P<0.001. C—H. Respective NOS expression, nNOS (C, F), iNOS (D, G) and eNOS (E, H), in HeLaX-E9s (C, D, E) and in HeLaX-Ss (F, G, H). Arrows in C, D, F, G and H point to tumor cells with pores (C, D) or without pores, and in E, fibrous cells; middle arrows in H, indicate DCs; small arrows in D, F and G point to presumptive immune cells, and in E and H, processes of DCs. Open arrows in C and E point to dying cells; open arrowheads in F point to immunopositive nNOS; and asterisks in D and H indicate remnant of dying cells. I—K. Corresponding regions to C, D and E stained with anti-cytochrome C indicating apoptotic death. Small arrows in I and long arrows in J and K point to tumor cells in apoptotic death. Open arrowheads in J point immune cells in apoptotic death. Asterisks in J, K indicate remnant of dying cells. L. Negative control for NOSs antibodies stained with rabbit IgG in HeLaX-E9-9s. Arrow points to tumor cells. M, N. Immunoblots for nNOS (M) and eNOS (N) proteins in HeLaX-E9s and their controls. Each band intensity is shown adjusted value by the density of β-actin in the HeLaX-Ss as 1.0. SM indicates safety margin of lung cancer which is the remainder used on 2010 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122458#pone.0122458.ref040" target="_blank">40</a>]. O. Localization of iNOS in macrophages (arrows) in HeLaX-E9-9s (a) and in NK cells (arrows) in HeLaX-E9-4.5s (b).