Effect of Id1 down-regulation on the growth of human leukemia cell lines. (A) Id1 expression in Molm-14 and K562 cells by Western blot analysis. The protein level of Id1 in stable antisense transfectant clones or transient si-RNA Id1 transfectant was expressed relative to that of parental cells by densitometry analysis. The membrane was stripped and reblotted with RACK1 antibody as loading controls, or reblotted with ID2 antibody as nonspecific controls. (B) Growth curve of Molm-14 and K562 cells measured by CellTiter 96 Aqueous One Solution Cell Proliferation Assay. Note that M1, M2 and Molm14-siId1 inhibit growth of Molm-14 cells, K1, K2, and K562-siId1 inhibit growth of K562 cells. Results shown are representative of 3 independent experiments. (C) Cell-cycle analysis of Molm-14 cells. Note that M1, M2, and Molm14-siId1 showed an increase in the G₁ population. Representative experiments are shown here. (D) Western blot analysis of Molm-14 and K562 cells using p27^Kip1 antibody. There is an approximately 2- to 3-fold increase of p27^Kip1 protein in antisense transfectant clones, compared with parental cells or cells transduced with pBabe empty vector. The membrane was stripped and reblotted with RACK1 antibody as loading control.