Figure 6

Modulation of LKB1 and SIK1 influences HTLV-1 proviral transcription. (A and B) LKB1 and SIK1 suppressed proviral transcription in a kinase-dependent manner. HeLa cells were co-transfected with pX1MT plus pCMV-Tag2 (vector), LKB1 (WT/D194A) or SIK1 (T182D/K56M). Cells were harvested 72 hrs after transfection. Total RNA was extracted and cDNA was synthesized. Semi-quantitative and quantitative RT-PCR was performed to analyze the relative levels of proviral Tax, Gag, Pol, Env, XII and GAPDH transcripts. The differences between bars 1 and 2, 1 and 4, 6 and 7, 6 and 9, 11 and 12, 11 and 14, 16 and 17, 16 and 19, 21 and 22, as well as 21 and 24 are statistically significant by two tailed Student’s t test (p values: 0.000014, 0.0026, 0.0036, 0.00061, 0.0033, 0.0075, 0.00031, 0.0078, 0.0000023 and 0.00013). (C) Verification of LKB1 knockdown in HEK293T cells. Cells were transfected with siRNAs against LKB1 or non-specific control siRNA (siNC) for 72 hrs. Cell lysates were analyzed by Western blotting. (D and E) Depletion of endogenous LKB1 potentiated HTLV-1 proviral transcription in infected cells. After 36 hrs of LKB1 knockdown, cells were co-transfected with pX1MT for 36 hrs. Cells were harvested and total RNA was extracted for cDNA synthesis. Semi-quantitative and quantitative RT-PCR was performed to analyze the expression of proviral Tax, Pol and Env transcripts. Statistically significant differences exist between bars 1 and 2, 1 and 3, 4 and 5, 4 and 6, 7 and 8, 7 and 9, 10 and 11, 10 and 12, 13 and 14, 13 and 15, 16 and 17, as well as 16 and 18 (p values: 0.000014, 0.00000078, 0.00000021, 0.0000012, 0.0000085, 0.00000014, 0.00000019, 0.00000060, 0.0000025, 0.000000071, 0.000035 and 0.000013).