0 × 103 cells/well). Cell viability was assessed by CCK-8 assay (Dojin Laboratories, Kumamoto, Japan). The absorbance at 450 nm ARS-1620 research buy (A450) of each well was read on a spectrophotometer. Three independent experiments were performed in quadruplicate. Western blotting Protein extracts from cell lines, patient samples prepared with RIPA lysis buffer (50 mM TrisHCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodiumdeoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0) were separated on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with an appropriate dilution (WT1 1:2000) of the primary antibody (Abcom, Cambridge, MA, USA),
followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (Abcom). The signals were detected by chemiluminescence phototope-HRP kit (Cell Signaling, Danvers, MA, USA). Blots were stripped and reprobed with anti-GAPDH antibody (Abcom) as an internal control. All experiments PX-478 purchase were repeated three times. siRNA, mimics, and anti-miR-15a/16-1 oligonucleotide (AMO) transfection SiRNA sequences targeting WT1: ccauaccagugugacuuca corresponds to positions
9-27 of exon 7 within the WT1 coding sequence. SiRNA-WT1 and unspecific control siRNA (N.C) were synthesized from Invitrogen. 50 nM SiRNA-WT1 or N.C were transfected into K562 and HL-60 cells using Hiperfect transfection reagent (Qiagen, Valencia, USA) according to manufacturer’s instructions. miR-15a or miR-16-1 mimics
was synthesized from Gene Pharma (Shanghai, China). 40 uM miR-15a or miR-16-1 mimics were transfected into K562 using Hiperfect transfection reagent (Qiagen). The sequences of AMO were designed according to the principle of sequences complementary to mature miRNA-15a/16-1. AMO and scramble (SCR) were chemically synthesized by Qiagen. AMO and SCR (final concentration of 50 nM) were transfected into K562 and HL-60 cells using the Hiperfect transfection reagent (Qiagen). All transfections were performed in triplicate for each time point. Statistical analysis The significance of the difference between Staurosporine chemical structure groups was determined by Student’s t-test. A P value of less than .05 was considered statistically significant. All Statistical analyses were performed with SPSS software (version 13). Results Pure curcumin downregulated the expression of WT1 and effectively inhibited cell proliferation in leukemic cells As reported previously , low concentration of pure curcumin could inhibit the growth of leukemic cells and downregulate the expression of WT1. The mRNA and protein levels of WT1 were detected by H 89 datasheet qRT-PCR and Western blotting respectively after K562 and HL-60 cells were treated with non-cytotoxic doses of pure curcumin (5, 10, 20 uM for K562 and 2.5, 5, 10 uM for HL-60) . As indicated in Figure 1A-D pure curcumin downregulated the expression of WT1 in time- and concentration -dependent manner.