rs under nor Crenolanib AML moxia, or grown under normoxia for 16 hours prior to exposure to hypoxia for 8 hours. After hypoxia, apoptosis was analyzed using Annexin V FITC PI binding staining and caspase 3 7 activity were mea sured by Cytomics FC500 flow cytometer. Total RNAs and protein were prepared for real time reverse transcription polymerase chain reaction and western blot analysis. RNA extraction and real time RT PCR Total RNA was extracted from cultured cells using Tri zol. The levels of mRNAs or miRNAs were measured by real time quantitative RT PCR using Bio Rad IQ5 system. For mRNA detection, reverse transcription was performed with Pri meScript RT reagent kit ac cording to the manufacturers instructions, and real time RT PCR was carried out using SsoFast EvaGreen Supermix kit with Bio Rad IQ5 real time PCR system.

The real time PCR reaction contained, 10 uL of SsoFast EvaGreen supermix, 1 uL of sense primer, 1 uL of anti sense primer, 2 uL of cDNA template, and 6 uL of H2O. The program of two step real time RT PCR was 95 C for 30 seconds, followed by 40 cycles of 95 C for 5 seconds, and 60 C for 10 seconds. The relative expres sion level of mRNAs was normalized to that of internal control B actin by using the 2 Ct cycle threshold method. Primer sequences were as follows, To detect the level of mature miR 494, the complementary DNA was synthesized using PrimeScript RT re agent kit and miRNA specific stem loop RT primers. The 10 uL of reaction contained, 2 uL of 5�� RT buffer, 0. 5 uL of Pri meScript RT Enzyme Mix, 1 uL of miR 494 RT primer, 1 uL of total RNA, and 5.

5 uL of H2O. The in cubation condition was 37 C for 15 minutes, followed by 85 C for 5 seconds. Then qRT PCR was performed with SsoFast EvaGreen Supermix kit and Bio Rad IQ5 real time PCR system. The reaction contained, 10 uL of SsoFast EvaGreen supermix, 1. 5 uL of forward primer, 1. 5 uL of reverse primer, 2 uL of cDNA template, and 5 uL of H2O. The program was the same as that described above. Forward and reverse primers were designed from RiboBio. U6 small nuclear RNA was used as an internal control. Protein extraction and western blot analysis Cells were washed twice quickly with ice cold phosphate buffered saline after either hypoxic or normoxic incubation, solubilized in 1�� lysis buffer with protease inhibitors and phosphatase inhibitors on ice.

Cell lysates were sonicated in an Ultrasonic Dismemberator GSK-3 on ice, followed by boiling for 5 minutes and selleck inhibitor centrifuging at 12000 g for 10 minutes at 4 C and the supernatants were retained. Protein con centration was determined by a BCA Protein Assay kit. For western blot, equal amounts of total protein in spe cial condition were loaded for electrophpresis in sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene fluoride microporous mem branes. After blocking for 1 hour at room temperature, the membranes were incubated with the primary antibodies overnight at 4 C. The fol lowing antibodies were used in this study, mo