(A) Immunofluorescence microscopy of TNIK (green) in U2OS cells. Nuclei (blue) were stained with DAPI. Scale bar: 20 μm. (B) Representative images of human OS tissues stained with anti-TNIK antibody. Rectangles indicate areas of magnification. Scale bar: 100 μm. (C) The 51 OS tissue samples were graded from 0 (no nuclear staining) to 5 (>75% of viable tumor cells) according to the percentage of tumor cells with nuclear TNIK expression. (D) Gene expression of TNIK (normalized to GAPDH) retrieved and analyzed from human OS data sets (19) in osteoblast (OB, n = 3), osteosarcoma (OS, n = 103), and mesenchymal stem cells (MSC, n = 12). The mean value of OB was set as 1. One-way ANOVA, **P < 0.01. (E) Blot intensity of TNIK (normalized to β-actin [loading control]) in the specimens before and after chemotherapy from good and poor responders. The sample after chemotherapy from case no. 3 was excluded from the analysis. Data represent the mean ± SEM. Mann-Whitney U test, *P < 0.05. PC, positive loading control. (F) Immunoblot analysis of TNIK and γ-tubulin (loading control) protein expression in osteoblast and OS cell lines.