LKB1 suppresses Tax-mediated LTR activation in a kinase-dependent manner. (A) Expression of LKB1 and Tax. HeLa cells were transfected with pIEX, pCMV-Tag2-LKB1, or pIEX plus escalating amounts of pCMV-Tag2-LKB1. Cell lysates were probed with mouse anti-Tax, mouse anti-Flag and mouse anti-α-tubulin (α-tub). (B) Kinase activity of LKB1 is required for suppression of Tax activity. HeLa cells were transfected with pLTR-Luc (100 ng), a fixed amount of pIEX (100 ng) and escalating amounts of LKB1 plasmids (25, 50 and 100 ng). Protein expression was analyzed (inset). Fold activation is calculated from pLTR-Luc activity normalized to that of pSV-RLuc. Results of this dual luciferase assay represent means from three independent experiments and error bars indicate SD. The differences between groups 2 and 6, and between groups 2 and 10 are statistically significant by two tailed Student’s t test (p = 0.00020 for both). In contrast, there is no statistically significant difference between groups 2 and 14 (p = 0.34) or between groups 2 and 18 (p = 0.29). (C) Kinase-dependent suppression of CRTC1 and Tax by LKB1. HeLa cells were transfected with pLTR-Luc (100 ng), a fixed amount of pIEX (100 ng) plus pCMV-Tag2-LKB1 (WT or D194A) (30 ng), and escalating amounts of expression plasmids (18, 37 and 75 ng) for CRTC1. Statistically significant differences are found between groups 4 and 8 (p = 0.00097) and between groups 16 and 20 (p = 0.000035). (D) LKB1 inhibition of LTR in T cells. Jurkat cells were transfected with pLTR-Luc (300 ng), a fixed amount of pCAG-Tax-V5 (200 ng) and escalating amounts of LKB1 plasmid (50, 100 and 200 ng). The difference between group 5 and groups 6–8 is statistically significant (p = 0.00084).