Metformin activates LKB1-SIK axis and suppresses HTLV-1 proviral gene expression and cell proliferation. (A) Treatment with metformin triggered phosphorylation of LKB1 and SIK1. Confluent HEK293T cells were exposed to metformin (0.5 and 1 mM for 30 min) as described . (B) Diminution of Tax protein in HTLV-1-transformed cells treated with metformin. Cells were treated with metformin (5, 10 and 20 mM) for 24 hrs. (C) Effect of metformin and 2-DG on Tax expression. MT4 cells were treated with metformin (10 mM) or 2-DG (50, 100, 150 mM) for 24 hrs. (D and E) Time course of metformin-induced downregulation of Tax mRNA production. Cells were treated with metformin (1 mM) for 24 and 36 hrs. Quantitative PCR was performed to analyze Tax and GAPDH transcripts. Relative Tax mRNA expression in mock-treated cells was taken as 1. *, p = 0.013. **, p = 0.004 (MT4 and MT2). ***, p = 0.00006. (F and G) Dose dependence of metformin-induced downregulation of Tax mRNA production. Cells were treated with metformin (2 and 5 mM) for 24 hrs. ***, p = 0.002 and p = 0.0003 for MT4; p = 0.0001 and p = 0.00003 for C8166. (H) Metformin inhibited HTLV-1 virion production. Cells were treated with metformin (5, 10 or 20 mM). Overnight production of cell-free HTLV-1 virions was assessed by measuring reverse transcriptase activity recovered from live HTLV-1 virus in the culture supernatant with a colorimetric assay kit (Roche). *, p = 0.0059. (I and J) Metformin inhibited proliferation of HTLV-1-transformed cells. Cell viability was measured by MTT method. (K) Metformin-mediated growth inhibition is LKB1-dependent. LKB1-depleted MT2 cells were treated with metformin (20 mM) for 24 hrs. **, p = 0.005, left; p = 0.008, right.