Silencing GLUT3 and PKM2 in hESCs

siRNA was used to silence expression of GLUT3 and PKM2 in Hues-7 hESCs cultured at 5% oxygen on Matrigel-coated plates. hESCs were transfected on the first day after passaging, before a standard medium change 24 hours later. Experiments were performed, or protein was collected, 48 hours after transfection. To silence GLUT3 expression, 50 nM of a previously validated siRNA (Ambion) was used with the transfection reagent INTERFERin (Polyplus). To silence PKM2 expression, 100 nM of a previously described PKM2 specific siRNA (Sense strand: 5′ CCAUAAUCGUCCUCACCAAUU 3′; Thermo Scientific)32 (link) was required, with the transfection reagent HiPerFect (Qiagen). Allstars control siRNA (Qiagen) was transfected into hESCs alongside GLUT3 siRNA and PKM2 siRNA experiments as a negative control.