A, B A549 cells were transfected with control siRNAs or siRNAs targeting HK1 or PKM2. After 2 days knockdown was confirmed by qPCR (A) and Western blot with GAPDH as loading control (B). Relative mRNA levels are shown (PKM2/PPIA and HK1/PPIA in % of levels in CT siRNA-transfected cells). Means of 3-5 experiments + SEMs. C HK1 siRNAs enhanced the cytotoxic effect of HAMLET in A549 cells. A549 cells were transfected with siRNAs targeting HK1, control siRNA or siRNAs targeting PKM2 and, after 2 days, treated with HAMLET (3 hours). ATP levels and viability in % of levels in respective untreated cells. Means of 3-5 experiments + SEMs. * for p < 0.05. D, E PFKFB1 siRNA decreased the cytotoxic effect of HAMLET in A549 cells. A549 cells were transfected with PFKFB1 siRNA or control siRNA for 48 hours and treated with HAMLET. D ATP levels in % of levels in respective untreated cells. Means of 3 experiments + SEMs. * for p < 0.05 E Knockdown of PFKFB1 was confirmed by qPCR and Western blot.