Figure 2

PAWI-2 affects KRAS-NF-κB signaling by targeting TBK1 phosphorylation to overcome tumor stemness. (A) Immunoblots and densitometry analysis of phospho-Ser172-TBK1 (pS172-TBK1) and TBK1 as determined with whole-cell extracts. (B–-E) TBK1 knockdown enhanced the effect of PAWI-2 in FG and FGβ₃ cells: (B) immunoblots show TBK1 genetic knockdown efficiency used in this study; effect of TBK1 knockdown (C) on cell viability inhibited by PAWI-2 as measured by a CellTiter-Glo assay and (D) effects on self-renewal capacity inhibited by PAWI-2 as measured by quantifying the number of secondary tumor spheres; (E) immunoblots and densitometry analysis of the effect of PAWI-2 on pS172-TBK1, TBK1, phospho-Ser403-p62 (pS403-p62), p62, phospho-Ser177-OPTN (pS177-OPTN), OPTN, or NDP52 in cells with TBK1 knockdown compared to control cells. (F,G) Enhancement of inhibition of (F) cell viability and (G) self-renewal capacity by co-treatment of PAWI-2 with TBK1 specific inhibitor (MRT67307, 1 µM). Concentrations of PAWI-2 used were as indicated: 50 nM in A, E, 10 nM in C, F and 20 nM in D, G; treatment time used was as indicated: 0–16 hours in A, 24 hours in C, D, F, G and 8 hours in E; vehicle control (0.5% DMSO). GAPDH or HSP90 was used as a loading control in A, B, E. Data are mean ± SD (n = 3) in C, D, F, G; P-values were estimated by Student t tests in C, D, F, G (*P < 0.05, **P < 0.01, ***P < 0.001). The full-length blots are presented in Supplementary Fig. S7.