Fig. 6. TRPM8 immunoscoring predicts X-rays + WS-12 efficacy.
a TRPM8 immunostaining of BM-18 PDX. Scale bars, 100 μm. b Western blotting analysis shows comparable expression levels of TRPM8 in BM-18 and RWPE-1 M8 cells. c Immunofluorescence images showing co-staining of Ki-67 (green, upper panel) or Cleaved Caspase-3 (green, lower panel) with CK8 (red) and DAPI (blue). d Percentage of Ki-67 positive cells on a total of 30,000 cells in at least five different areas of the sample. Scale bars, 50 μm. e Western blotting analysis in BM-18 PDX tissues slices upon WS-12 (1 μM, 48 h), X-rays (10 Gy), or X-ray + WS-12 treatments showing molecular hallmarks of apoptotic cell death (Caspase-3 and PARP cleavage). Error bars, mean ± SD. Data were analyzed using a two-tailed Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. f Representative flow cytometry analysis of apoptotic cell death by Annexin-V/Sytox-Green labeling in LNCaPFGC M8 cells treated with WS-12 (1 μM), docetaxel (5 nM), enzalutamide (1 μM), WS-12 + docetaxel, or WS-12 + enzalutamide for 48 h. Untreated cells were used as control. g Quantification of dying cells in LNCaPFGC expressing endogenous (WT), increased (M8) or knocked-out (CAS) levels of TRPM8 treated as indicated in f. h Western blotting analysis of the indicated samples showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of LNCaPFGC WT and M8 cells and the molecular signature of apoptotic cell death (Caspase-3 and PARP cleavage) upon treatment with combination of WS-12 with docetaxel or enzalutamide. Error bars, mean ± SD. Experiments were performed in quadruplicate; data were analyzed using a two-way ANOVA test. **P ≤ 0.01.