Requirement of SIKs, CRTCs and CREB in LKB1-mediated suppression of HTLV-1 LTR. (A) Compromising CRTC1 and CREB dampened LTR activity in LKB1-depleted cells. LKB1-depleted HEK293T cells, as shown in Figure 3A, were cotransfected with plasmids pLTR-Luc, pSV-RLuc, pIEX, pM1-CRTC1 and pA-CREB. * and #, the difference between the two groups is statistically significant by two tailed Student’s t test (p = 0.020 and p = 0.0014). (B) Verification of AMPK knockdown in HeLa cells. RT-qPCR was performed to analyze AMPK and GAPDH transcripts. The difference between bars 1 and 3 or 2 and 4 is statistically significant (p = 0.00036 or 0.0018). (C) Verification of SIK knockdown in HeLa cells. p values were obtained for selected bars (1 and 4: 0.00046; 2 and 5: 0.0046; 3 and 6: 0.00011). (D) Endogenous SIKs in HeLa cells are required for LKB1-mediated LTR suppression. AMPKs and SIKs were targeted by independent siRNAs at a concentration of 100 nM. siGFP was used as a negative control. After 36 hrs of knockdown, plasmids pLTR-Luc, pSV-RLuc, pIEX and pCMV-Tag2-LKB1-WT were cotransfected into cells. Cells were harvested 36 hrs after the second transfection. Statistically significant differences exist between group 3 and each of groups 5–11 (p values: 0.041, 0.00042, 0.0057, 0.00028, 0.00021, 0.0018 and 0.0017), whereas the difference between groups 3 and 4 was insignificant (p = 0.735). (E) Phosphorylated CRTC1 is required for LTR suppression. HeLa cells were transfected with pLTR-Luc reporter (100 ng), a fixed amount of Tag2-SIK2-T175D (100 ng) or Tag2-SIK3-T163D (100 ng), and escalating amounts of CRTC1 plasmid (18, 37 and 75 ng). Cells were harvested 36 hrs after transfection. The difference between groups 16 and 20 or 16 and 24 is statistically significant (p = 0.00054 or 0.00038).