Knockdown of HK2 as well as miR-143 overexpression results in decreased lactate secretion. A, Verification of HK2 knockdown on protein level. Cells were transfected on two consecutive days with HK2 siRNA and protein was harvested 48 h after the first transfection. Tubulin was used as loading control. B, Verification of HK2 knockdown on transcript level. DLD-1 cells were transfected on two consecutive days with HK2 siRNA and RNA was harvested 48 h after the first transfection. The expression level was normalized to ATCB and shown as the mean ± S.D. of three replicates. C, HK2 knockdown results in a reduced proliferative potential of DLD-1 cells. DLD-1 cells transfected with miR-143 duplex or HK2 siRNA exhibit a reduced cell proliferation as measured by crystal violet growth assay. Results are shown for day 4 after transfection as the mean ± S.D. of four replicates. *, p < 0.005 using a two-tailed t-test. D, Lactate secretion of DLD-1 cells is decreased upon miR-143 overexpression or HK2 knockdown compared to mock transfected cells. Cells were transfected as described in A) and B) and media samples were removed in 6 h intervals for measurement of lactate. The 0 h time points correspond to samples taken immediately after addition of fresh media following the second transfection. Data shows a representative experiment with results depicted as the mean ± S.D. of three replicates. *, p < 0.005 using a two-tailed t-test.