(A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), colorimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G₁ fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O₂ = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the preculture, and subjected to the clonogenic survival assay. (H–K) The indicated cells were transfected with either pcDNA4/SPINK1-ΔSP (SPINK1-ΔSP) or its EV and cultured for 48 hours. Then, both the culture media and cell lysates were subjected to Western blotting using the indicated antibodies (H and J), and then, cells were irradiated with the indicated doses of γ-rays and subjected to the clonogenic survival assay (I and K). The exogenously expressed SPINK1-ΔSP was detected using anti-myc tag Ab (H and J). (L) After 24 hours serum starvation, DU145 cells were treated with or without 100 ng/mL rSPINK1 in combination with SPINK1-neutralizing antibody or control IgG (0.5 μg/mL) for 24 hours and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in C–E and L, and n = 6 in B, F, G, I, and K). Student’s t test. *P < 0.05, **P < 0.01. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.