The knockdown of the M2 isoform of pyruvate kinase mRNA by si27, si155, and si156 is potent and specific. (A) HCT116 cell were transfected with 5 nM siRNA. Duplicate biological samples were collected 48 h after transfection. Quantitative real-time PCR primers were designed to amplify total PKM, PKM1 only, or PKM2 only from HCT116 total RNA. HCT116, HepG2, and SKOV3 cells were transfected with 5 nM siRNA at 0, 48, and 96 h. (B and C) Cells were assayed for cell viability (B) and apoptosis (C) after 6 d. Results are normalized relative to the no treatment control. Transfections were performed in technical triplicate on two independent occasions. NT, no treatment. siPK, siRNA targeting both M1 and M2 isoforms. siControl, siRNA targeting firefly luciferase. Error bars denote standard deviation. (D) HCT116 cells were transfected with serial dilutions of si156 at 0, 48, and 96 h. Cells were assayed for cell viability after 6 d. Results are normalized relative to the no treatment control. Transfections were performed in technical quadruplicate on two independent occasions. Error bars denote standard deviation. (E) Lysates were collected from HCT116, HepG2, and SKOV3 cells 48 h after transfection with 5 nM siRNA. Blots were probed with antibodies targeting either total PKM or PKM2. Duplicate biological samples were collected for HepG2 and SKOV3, the cell lines used in the xenograft experiment. NT, no treatment. Vinculin was used as a loading control. (F) Effect of si156 on lactate production in HCT116 cells. The assay was performed 48 h after cells were transfected with 5 nM siRNA. Results are normalized to the no treatment control, and lactate production is further normalized to cell number. The experiment was performed in biological and technical duplicate on two independent occasions. Error bars denote standard deviation.