(A) Effect of glucose deprivation on miR-451 expression levels. Cells were cultured for 24 hr in normal (+, glucose 4.5 g/l) or reduced glucose (−, glucose 0.3 g/l) medium. miR-451 levels were then measured by qRT-PCR and expressed as mean relative to normal medium controls ± SD.
(B) Effect of AMPK and LKB1 downregulation on miR-451 expression. U87, U251,and U373 glioma cells were transfected with negative control siRNA, AMPKα1 siRNA, or LKB1 siRNA. miR-451 levels were measured 48 hr after transfection by qRT-PCR and expressed as mean relative to controls ± SD (left). Corresponding western blots with anti-AMPK, anti-LKB1, and anti-α-tubulin antibody as a loading control are shown below (right).
(C) Effect of miR-451 expression on glucose deprivation-dependent AMPK activation. U251 cells were transfected either with negative control miR (NC) or miR-451 precursor and grown in normal (+, glucose 4.5 g/l) or reduced glucose (−, glucose 0.3 g/l) medium for 18 hr. Cell lysates were blotted with anti-CAB39, anti-phospho-AMPK, anti-AMPK, anti-phospho-Raptor, and anti-Raptor antibodies. Anti-α-tubulin antibody was used as a loading control.
(D) Effect of miR-451 expression on cellular response to glucose deprivation. U251 cells were transfected either with negative control miR (NC) or miR-451 precursor and were grown in normal (+, glucose 4.5 g/l) or reduced glucose (−, glucose 0.3 g/l) medium for 24 hr. Cell number was quantified by crystal violet staining and was expressed as mean cell number relative to controls ± SD. *p < 0.01. See also Figure S3.