A549 cells were plated onto 6-well plates one day prior to transfection. Following confirmation of 70%–80% confluence, the cells were transfected with {Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|buy Anti-diabetic Compound Library|Anti-diabetic Compound Library ic50|Anti-diabetic Compound Library price|Anti-diabetic Compound Library cost|Anti-diabetic Compound Library solubility dmso|Anti-diabetic Compound Library purchase|Anti-diabetic Compound Library manufacturer|Anti-diabetic Compound Library research buy|Anti-diabetic Compound Library order|Anti-diabetic Compound Library mouse|Anti-diabetic Compound Library chemical structure|Anti-diabetic Compound Library mw|Anti-diabetic Compound Library molecular weight|Anti-diabetic Compound Library datasheet|Anti-diabetic Compound Library supplier|Anti-diabetic Compound Library in vitro|Anti-diabetic Compound Library cell line|Anti-diabetic Compound Library concentration|Anti-diabetic Compound Library nmr|Anti-diabetic Compound Library in vivo|Anti-diabetic Compound Library clinical trial|Anti-diabetic Compound Library cell assay|Anti-diabetic Compound Library screening|Anti-diabetic Compound Library high throughput|buy Antidiabetic Compound Library|Antidiabetic Compound Library ic50|Antidiabetic Compound Library price|Antidiabetic Compound Library cost|Antidiabetic Compound Library solubility dmso|Antidiabetic Compound Library purchase|Antidiabetic Compound Library manufacturer|Antidiabetic Compound Library research buy|Antidiabetic Compound Library order|Antidiabetic Compound Library chemical structure|Antidiabetic Compound Library datasheet|Antidiabetic Compound Library supplier|Antidiabetic Compound Library in vitro|Antidiabetic Compound Library cell line|Antidiabetic Compound Library concentration|Antidiabetic Compound Library clinical trial|Antidiabetic Compound Library cell assay|Antidiabetic Compound Library screening|Antidiabetic Compound Library high throughput|Anti-diabetic Compound high throughput screening| pGL3-Basic without promoter (negative control), pGL3-SVP-229-luc (mutant plasmid), and pGL3-SVP-230-luc (normal plasmid). For cell transfection, A549 cells were transiently transfected with 2 μg plasmids buy BIX 1294 and 0.2. g internal control plasmid pRL-TK by using Lipofectamine 2000™ reagent according to the manufacturer’s instructions. Luciferase reporter gene expression detection Thirty hours after transfection, cells were harvested and lysed with 1 × lysis buffer (Promega),

and then 20 μl of cell extract was assayed for luciferase activity using the Dual-Luciferase assay kit (Promega) according to the manufacture’s instructions. The relative level of reporter gene expression was expressed as the ratio of firefly luciferase activity to Renilla luciferase (LU/RL). RNA interference A double strand siRNA oligonucleotide targeting HIF-1α (sense: 5-CUGAUGAC CAGCAACUUGAdTdT-3, antisense: 5-UCAAGUUGCUGGUCAU CAGdTdT-3) was designed based on the reference [21] and synthesized by Shanghai Genepharma Co. Ltd. (China). A pair of negative control siRNA were also designed with sequences

different from siRNA-HIF-1α and not homologous to any sequences found in gene bank (sense: 5-AGUUCAACGACCAGUAGUCdTdT-3, antisense: 5-GACUACUGGUCGUUGA Hedgehog inhibitor dTdT-3). For transfection, cells were plated onto 10 cm2 cell culture dishes and grown to 30–50% confluence

before transfection. Bay 11-7085 50 μl of Oligofectamine transfection reagent per dish (Invitrogen) was added, and the cells were incubated at room temperature for 20 min. The cells were then rinsed with Opti-Mem I to remove any residual serum. The siRNA duplexes were diluted to a final concentration of 20 nM in Opti-Mem I (Invitrogen). Cells were incubated with the oligonucleotide duplexes in serum-free conditions for 4 h at 37°C. Serum was then added back to the culture, and cells were incubated in normoxic or hypoxic condition for an additional 48 h. Real Time Reverse Transcription-PCR Total RNAs were isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Twenty-five nanogram total RNA per sample was reverse transcribed by using the Reverse Transcription Reaction Kit (Takara Code: DRR061S) according to the manufacturer’s instructions. Quantitative real-time PCR was performed analyzed on the Applied Biosystems 7300 Real-Time PCR System to determine the relative amounts of survivin, HIF-1α and GAPDH (internal control) mRNAs expressed. The SYBR Green Supermix was used for all real-time PCR reactions.