Effects of Lyn, Btk, and Hck siRNA of the viability and proliferation of HMC-1.2 cells. (A-E) HMC-1.2 cells were kept in control medium or transfected with siRNA directed against luciferase (Luc siRNA, 200nM) or siRNA against Lyn, Btk, or Hck (100-200nM each) using lipofectin as described in “Design and application of siRNA.” After 16 hours, cells were examined for Lyn, Btk, and Hck protein expression by Western blotting using Abs against Lyn, Btk, Hck, and β-actin (loading control; A). Cell viability and apoptosis in each condition were determined after 16 hours by light microscopy (B), combined annexin V/propidium iodide staining after 48 hours (C), or by TUNEL assay after 16 hours (D). Cell proliferation after 24 hours was determined by 3H-thymidine uptake (E). (F-G) HMC-1.2 cells were transfected with control siRNA (Luc siRNA) or 100nM Lyn or Btk siRNA as indicated. Four hours after transfection, cells were washed and resuspended in IMDM containing 10% FCS. Then, cells were kept for 24 hours in control medium or in the presence of 100nM midostaurin. Proliferation was determined by 3H-thymidine uptake (F), and the numbers of apoptotic cells by light microscopy (G). Results represent the mean ± SD of 3 independent experiments. *P < .05.
