Knockdown of LKB1 enhances Tax expression in HTLV-1-transformed cells. (A) Expression of LKB1 and SIKs in HTLV-1-transformed cells. RT-qPCR was performed to analyze LKB1, SIK1/2/3 and GAPDH transcripts. Quantitation of mRNA expression was achieved by comparative Ct method. The differences between bars 5 and 6, 5 and 7, 5 and 8, as well as 13 and 16 are statistically significant by two tailed Student’s t test (p values: 0.000011, 0.000082, 0.000000076 and 0.00040). (B and C) Verification of LKB1 knockdown by siRNA. siLKB-1 was transfected into MT4 and C8166 cells using TransIT-Jurkat transfection reagent (Mirus). Endogenous proteins were analyzed by Western blotting at 48 hrs post-transfection using mouse anti-LKB1, mouse anti-Tax and mouse α-tubulin (α-tub). Relative amounts of LKB1 or Tax normalized to α-tubulin were determined by densitometry and are indicated at the bottom of the panels. (D and E) Knockdown of endogenous LKB1 enhanced Tax expression in HTLV-1-transformed cells. siLKB1-1 at a concentration of 100 nM was used to deplete endogenous LKB1 in MT4 and C8166 cells. Cells were harvested and total RNA was extracted for cDNA synthesis. Quantitative PCR was performed to analyze Tax and GAPDH transcripts. ***, the difference between the two groups is statistically significant by two tailed Student’s t test (p = 0.00005, MT4; p = 0.001, C8166).