Article Title: Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3
Figure Lengend Snippet: Reelin promotes the activation of Syk and STAT3 via integrin β1 A. Reelin induces the activation of Syk and STAT3. H929 cells transfected with pCrl or control plasmid were cultured in 5% BSA- or FN-coated plates for 1 hour. The cells were then subjected to western blotting with phospho-STAT3 (Tyr705), total STAT3, phospho-Syk (Tyr525/526), and total Syk-specific antibodies. B. Densitometric quantification of phospho-STAT3 over total STAT3 from the blot shown in (A). C. Reelin promotes activation of Syk and STAT3 in H929 cells treated with Doxorubicin. H929 cells were transfected with pCrl or reelin-specific siRNAs for 40 hours and were then treated with Dox (2 μM) in the presence or absence of FN. Twenty-four hours later, the cells were subjected to western blotting for the activation of Jak2 (phospho-Jak2, Tyr1007/1008), Syk, and STAT3. D. Densitometric quantification of phospho-STAT3 over total STAT3 from the blot shown in (C). E. The reduced activation of FAK, Src, Syk, and STAT3 caused by reelin-specific siRNAs is ameliorated by the addition of recombinant reelin. Reelin-specific siRNA-transfected H929 cells were treated with Dox in FN-coated wells in the presence of BSA or recombinant reelin. Cell harvesting and western blotting was performed 24 hours later. F. The effects of integrin β1 inhibitory antibody, Src inhibitor, Syk inhibitor and PI3K inhibitor on the activation of β1 signaling pathway. H929 cells were transfected with pCrl or control plasmid. Forty hours later, the cells were cultured in FN-coated plates and were treated with integrin β1 inhibitory antibody P4C10, Src inhibitor PP2, Syk inhibitor BAY 61-3606, or PI3K inhibitor LY 294002 for 24 hours. The cells were then lysed and subjected to western blotting. Data are representative of three independent experiments.
Article Snippet: Immunoblotting and confocal microscopy Following incubation under the indicated conditions, including the addition of integrin β1-blocking antibodies (20 μg/ml), control mouse IgG antibodies (20 μg/ml), Src inhibitor PP2 (1 μmol/L, Merck Millipore), Syk inhibitor IV, BAY 61-3606 (1 μmol/L, Merck Millipore), PI3K inhibitor LY 294002 (50 μmol/L, Cell Signaling Technology), MM cells were collected and washed twice with ice-cold PBS, and incubated for 10 minutes at 4°C in Triton X-100 lysis buffer (30 mM Tris-HCl pH7.5, 150 mM NaCl, 25 mM NaF, 1% Triton X-100, 10% glycerol, 2 mM Naorthovanadate).
Techniques: Activation Assay, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Recombinant, Cell Harvesting