The experiment was repeated three times independently. Chromatin immunoprecipitation (CHIP) After fixed with 1% formaldehyde, AML cells were treated with ultrasonication. HOXA5 (ab82645, 1:500), Bcl-2 Associated X (Bax, ab32503, 1:1000), Bcl-2 (ab32124, 1:500), MCP-1 (ab9669, 1:500), cleaved-caspase3 (ab49822, 1:500), p27 (ab32034, 1:5000), and cyclin G (ab170389, 1:100) at 4?C overnight. After being washed three times with phosphate buffered saline-Tween 20 (PBST), the membranes were incubated after the addition of horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (ab205719; 1:2000, Abcam Inc., Cambridge, MA, USA) at room temperature for 1?h. Then the membranes were washed three times with PBST and detected using the enhanced chemiluminescence (ECL, EMD Millipore Corporation, Billerica, MA, USA). Gray-value quantification of bands in western blot images was performed using Image J analysis software, and GAPDH was taken as an internal reference. The experiment was repeated SK1-IN-1 three times. Fluorescence in situ hybridization (FISH) The location of HOTAIR in AML cells was detected by FISH according to the instructions of RiboTM lncRNA FISH Probe Mix (Red) (Guangzhou RiboBio Co., Ltd., Guangzhou, China). AML cells were cultured in 6-well plates, which were coated with coverslips for 1 d until the cell confluence reached about 80%. After that, cells were washed with phosphate-buffered saline (PBS), fixed with 1?mL of 4% paraformaldehyde at room temperature. After being treated with 2?g/mL protease K, glycine and acetylation reagents, cells were incubated with 250 L of pre-hybridization solution at 42?C for 1?h. After the pre-hybridization solution was aspirated, cells were added with 250?L of hybridization solution containing 300?ng/mL HOTAIR probe and then hybridized overnight at 42?C. After washing with PBST three times, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:800) diluted with PBST for 5?min, rinsed with PBST three times (3?min each time) and sealed with anti-fluorescent quencher. Five Mouse monoclonal antibody to Hsp70. This intronless gene encodes a 70kDa heat shock protein which is a member of the heat shockprotein 70 family. In conjuction with other heat shock proteins, this protein stabilizes existingproteins against aggregation and mediates the folding of newly translated proteins in the cytosoland in organelles. It is also involved in the ubiquitin-proteasome pathway through interaction withthe AU-rich element RNA-binding protein 1. The gene is located in the major histocompatibilitycomplex class III region, in a cluster with two closely related genes which encode similarproteins different SK1-IN-1 fields were selected and photographed under a fluorescence microscope (Olympus Co., Ltd., Tokyo, Japan). Each experiment was repeated three times. Methylation specific PCR (MS-PCR) Based on the DNA Methylation-Gold? Kit (D5005, Zymo Research, Irvine, CA, USA), the methylation level of the HOXA5 promoter region was measured. The primer sequences for methylation reaction were HOXA5-MD (5-TTTAGCGGTGGCGTTCG-3) and HOXA5-MR (5-ATACGACTTCGAATCACGTA-3), and the primer sequences for the un-methylation reaction were HOXA5-UD (5-TTGGTGAAGTTGGGTG-3) and HOXA5-UR (5-AATACAACTTCAAATCACATAC-3). The purified DNA was added into cytosine to thymine (CT) conversion reagent for denaturation and bisulfite conversion. Then the desulfurization and purification were conducted using a reaction column, and the purified DNA could be used for subsequent PCR reaction. The PCR reaction conditions were as follows: pre-denaturation at 95?C for 10?min, and 35 cycles of denaturation at 95?C for 45?s, methylation at 56?C for 45?s, non-methylation at 45?C for 45?s, extension at 72?C for 45?s, and a final elongation at 72?C for 10?min. The reaction products subsequently underwent agarose gel electrophoresis, which were then analyzed by imaging analysis. Each experiment was repeated three times. Dual luciferase reporter assay HOXA5 promoter region was detected by dual luciferase reporter assay. Cells were inoculated into the 24-well plates and cultured with plasmids using Lipofectamine 2000 when cells confluence reached 60C80%. The cells were collected after 24C48?h, rinsed with PBS three times and lysed with 75?L lysate at SK1-IN-1 room temperature for 15C20?min, shaken every several min so that SK1-IN-1 the cells could be completely covered with lysate. After collection of the cell lysate, luciferase activities were immediately detected based on the instructions of Dual luciferase assay kit with a luminometer (Monolight 2010; SK1-IN-1 Analytical Luminescence Laboratory, San Diego, CA, USA). During the experiment,.