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. 2021 Feb 8;6(3):e137245. doi: 10.1172/jci.insight.137245

Figure 4. TNIK inhibition abrogates OS stemness.

Figure 4

(A and B) Anchorage-independent colony formation by OS cells. U2OS cells were embedded in soft agar; covered by media containing DMSO alone (control), 1 μM NCB-0846, or 1 μM NCB-0970; and cultured for 14 days. (A) Representative images of colonies are shown (scale bar: 100 μm). (B) Relative cell viability was determined colorimetrically at 450 nm (n = 4). Data represent the mean ± SD. (C) Frequency of sphere formation by OS cells. U2OS cells were treated with DMSO, 1 μM NCB-0846, or 1 μM NCB-0970 for 3 days. The frequency of sphere formation and statistical significance were calculated using Extreme Limiting Dilution Analysis (ELDA) software. **P < 0.01. (D) Immunoblot analysis of TNIK and transcription factors related to cancer stemness (SOX2, NANOG, OCT4A, and MYC) in U2OS cells treated with DMSO, 1 μM NCB-0846, or 1 μM NCB-0970 for 72 hours. (E) TNIK knockdown suppresses the sphere-forming activity of OS cells. USO2 cells stably expressing shCtrl (Ctrl) or shTNIK (#2) were distributed into 96-well U-bottomed culture clusters at a density of 1, 10, or 100 cells per well and cultured for 14 days. The frequency of sphere formation and statistical significance were calculated using the ELDA software. *P < 0.05. (F) Immunoblot analysis of TNIK, proteins related to cancer stemness (SOX2, NANOG, OCT4A, and MYC), and γ-tubulin (loading control) in U2OS cells stably expressing shCtrl or shTNIK clone 33 (#1) and clone 35 (#2). (G) Expression of the SOX2, NANOG, OCT4, and MYC genes (normalized to ACTB) in NOS-10 xenografts excised from immunodeficient mice administered water (vehicle, n = 10) or 80 mg/kg NCB-0846 HCl (n = 10). Student’s t test, **P < 0.01, ***P < 0.001. Data represent the mean ± SEM.